Stabilisation of nucleic acids

ABSTRACT

The present invention concerns a method for the stabilisation of nucleic acid from a biological sample, which comprises: (a) collecting of biological sample; (b) treating the sample so that a proportion of the 2′, 3′ or 5′-OH positions of the nucleic acid are modified with a protecting group; and (c) subjecting the treated sample to one or more steps to isolate nucleic acid therefrom; wherein the modified nucleic acid is subjected to a deprotection step comprising treatment with a primary amine to remove the protecting group.

FIELD OF THE INVENTION

The present invention relates to a method for the stabilisation andpartial or complete isolation of nucleic acids including DNA and RNA.

BACKGROUND TO THE INVENTION

Various methods are known for the purification of nucleic acids such as(i) the use of a salt chaotrope and silica surfaces, (ii) a phenol basedextraction, and (iii) a chaotrope and precipitation of the nucleic acid.Methods for the purification of nucleic acids have been extensivelydescribed (Sambrook et al., (1989) Molecular Cloning: A LaboratoryManual, CSH).

These methods do not provide a method to stabilise the analyte nucleicacid prior to its extraction, rather the nucleic aicds are extracted asquickly as possible from the sample in order to minimise degradation.Other drawbacks of these methods are that the purified nucleic acids arecontaminated with proteins and other biomolecules that lead toinhibition of downstream enzymatic applications and possible cleavage ofthe nucleic acid. Many of the methods are also time consuming and resultin loss of a significant proportion of the desired nucleic acid analyte.Indeed one of the downsides of obtaining higher purity nucleic acid isthat the overall yield of the nucleic acid is reduced.

SUMMARY OF THE INVENTION

The present invention aims to overcome disadvantages in the prior art.

Accordingly, in a first aspect, the present invention provides a methodfor the stabilisation of nucleic acid from a biological sample, whichcomprises:

(a) collecting a biological sample;

(b) treating the sample so that a proportion of the 2′, 3′ or 5′-OHpositions of the nucleic acid are modified with a protecting group; and

(c) subjecting the treated sample to one or more steps to isolatenucleic acid therefrom;

wherein the modified nucleic acid is subjected to a deprotection stepcomprising treatment with a primary amine to remove the protectinggroup.

In a second aspect, the present invention provides a kit for use in amethod for the stabilisation of nucleic acid from a biological sample,which comprises:

(i) a reaction system for treating the sample so that a proportion ofthe 2′, 3′ or 5′-OH positions of the nucleic acid are modified with aprotecting group;

(ii) an isolation system for subjecting the treated sample to one ormore steps to isolate nucleic acid therefrom; and

(iii) a primary amine for subjecting the modified nucleic acid to adeprotection step to remove the protecting group.

In a further aspect of the invention there is provided a method for theisolation of a nucleic acid from a biological sample which comprises:

-   -   (a) collecting a biological sample;    -   (b) binding the nucleic acid to a solid phase in the present of        an organic solvent;    -   (c) optionally washing the solid phase to remove contaminants;        and    -   (d) eluting the nucleic acid from the solid phase; wherein the        solid phase contains a metal or metal ion capable of        coordinating with phosphate. In this aspect of the invention,        the sample may or may not have been treated so that a proportion        of the 2′, 3′ or 5′-OH, positions thereof are modified with a        protecting group. Where this treatment has been made, the        nucleic acid may be deprotected either before or after binding        to the solid phase. The nucleic acid may be eluted from the        solid phase using a chelator which may be subsequently removed        using ultrafiltration, photosensitivity (where the chelator is        photosensitive) or by affinity purification using an affinity        tag on the chelator.

The proportion of 2′, 3′ or 5′-OH positions modified depends on the formand type of the nucleic acid to be modified. Whilst RNA contains 2′, 3′and 5′-OH groups, DNA bears only 3′ and 5′-OH groups. For RNA, aproportion is defined as at least one modification of the 2′ positions,more preferably at least 10%, even more preferably at least 50%, evenmore preferably 75% and most preferably 100%. It is expected that forRNA modified at 5% or more of the 2′-OH positions, that at least one ofthe 3′ or 5′ hydroxyl positions will also be modified per molecule butthis will also depend on whether the RNA bears a 5′phosphate rather thana 5′-OH group in which case the proportion will be lower. For DNA, aproportion can be defined as at least one 3′ or 5′-OH group modificationper molecule where the molecule can be either single or double strandedDNA.

The solid phase is preferably hydroxylapatite, in which calcium ions arepresent. Other solid phases include iron (3) oxide and commerciallyavailable nickel-containing beads. Suitable metals include sodium,lithium, potassium, cesium, magnesium, titanium, chromium, manganese,calcium, iron, cobalt, nickel, copper, zinc, aluminium, silver, gold,platinum and lead, a metal oxide, a mixture of metals such as aniron-zinc blend or an oxide thereof, lithium iron III oxide, and ionsthereof. The organic solvent is preferably water miscible and may be anyone of the organic solvents described herein.

Suitable solvents include; tetrahydrofuran, dioxolane,N-methylpyrrolidinone (NMP), Formyl morpholine, Dimethyl imidazolidone,acetoxy acetone and acetonyl acetone. In a further aspect, the presentinvention provides a method for the stabilisation of nucleic acid from abiological sample, which comprises:

(a) collecting a biological sample;

(b) treating the sample so that a proportion of the 2′, 3′ or 5′-OHpositions of the nucleic acid are modified with a protecting group; and

(c) subjecting the treated sample to one or more steps to isolatenucleic acid therefrom;

wherein step (b) is carried out in a reaction medium which comprises anorganic solvent having a flashpoint above 37° C., preferably above 60°C. and more preferably above 90° C., which solvent is capable of forminga homogeneous solution with human blood when mixed in a ratio of 5:1(vol:vol). Preferred solvents include N-methylpyrrolidinone, formylmorpholine and dimethyl imidazolidone.

In a further aspect, the present invention provides a method forimproving the template activity of a nucleic acid which comprisestreating the nucleic acid with a metal ion chelator such as EDTA or EGTAto remove therefrom substantially all metal ions. Template activityincludes reverse transcription using a reverse transcriptase such asAMV, MULV or TTh DNA polymerase or DNA polymerisation using a DNApolymerase such as Taq, Tth or Klenow fragment or RNA polymerisationusing an RNA polymerase such as T3, 17 or SP6. The chelator is thenremoved from the nucleic acid by for example, ultrafiltration and thenucleic acid added to the polymerase reaction mixture whereupon themetal ions in the reaction bind to the phosphate of the nucleic acid. Ithas been found that nucleic acids treated in this manner have greatlyimproved template activity.

We have developed a method that provides nucleic acids of high purityand yield, but also in the case of RNA and DNA, are chemically protectedfrom degradation prior to extraction thereby improving analyticalprecision. We have found that surprisingly, 2′-OH RNA is not onlyprotected from nuclease, divalent metal cation and alkali degradationbut also from freeze-thaw promoted degradation which is useful for RNAcontrols and standards which may be refrozen several times after analiquot is used.

Described is a method for purifying DNA and RNA from a sample, such as abiological sample or a clinical sample including cells, blood, serum andplasma. Advantageously, when the analyte is RNA, it is protected fromdegradation by chemical modification of the 2′-OH groups. The chemicallyprotected RNA can be consequently deprotected using an organic primaryamine which have been found to be particularly suited for this purpose.For both DNA and RNA purification, the method provides a means tostabilise nucleic acids in the sample, lyse cells and virus particlesand remove proteins. Elution from the solid phase hydroxylapatitepurification matrix is effectuated using a metal ion chelator such asEGTA.

Deprotection of Nucleic Acids

Part of the invention relates to methods to remove acyl groups such asacetyl groups from acylated molecules such as 5′ and/or 3′ modified DNAand 2′ modified RNA. It is known that many if not all methods that havebeen described to remove, for example acetyl groups from acetylatedcompounds can lead to non-desired products along with the desirednon-acetylated product. This is because the nucleic acid can be cleavedby the deprotecting compound. In this case the deprotecting compound isdefined as a material that can remove an acyl group and in particularacetyl groups from an oxygen (acetyl) or nitrogen (acetamide) to restorethe original hydroxyl or amide group respectively with only limiteddegradation of the nucleic acid itself. One of the constraints ofremoving acyl groups from RNA and DNA polymers compared witholigonucleotides is the increased probability of chain cleavage. Anaturally occurring RNA polymer such as mRNA and viral RNA are onaverage 2,000-10,000 nucleotides in length. According to the presentinvention an oligonucleotide generally has a sequence length of up toabout 80 bases and a polynucleotide generally has a sequence length ofmore than about 80, preferably more than about 100 bases. A preferredlength for a polynucleotide is at least 1,000 bases. In order to removeacyl groups from a polymer compared with an oligonucleotide istechnically difficult because of the increased risk that there will alsobe a non desired cleavage of the sugar-phosphate backbone. Degradationcan be defined as cleavage of the sugar-phosphate backbone so that the5′ and 3′ ends of the nucleic acid become separated and is particularlya problem when handling RNA. For a 2′-OH acetylated RNA molecule of, forexample, 10,000 nt in length, it is desired that the number of sugarphosphate cleavages occuring during removal of the acetyl groups is lessthan 5%, more preferably less than 1%, even more preferably less than0.01%.

The ease with which the acyl group can be removed from the acylatedcompound depends on a number of parameters that have been describedextensively in <<Protecting Groups in Organic Synthesis>> Greene andWuts, 2^(nd) Edition, Wiley Interscience. In general, acyl groups areremoved more easily from oxygen than from nitrogen. Another major factoris the group attached to the carbonyl, for example the formyl group(—CO—H) is simply removed at pH 9 and above, whilst the acetyl group(—CO—CH3) requires much harsher conditions to remove it. Longer chainlengths such as propanoyl (—CO—CH2-CH3) and butanoyl (—CO—CH2-CH2-CH3)are even more difficult to remove whilst substituted acyl groups such asmethoxyacetyl (—CO—CH2-O—CH3), chloroacetyl (—CO—CH2Cl) ortrifluoracetyl (—CO—CF3) are much more readily removed than theunsubstituted acetyl. However, acetyl remains the predominately usedacyl protecting group throughout industry, in part because it can bereadily added to compounds from cheap and easily used reagents such asacetic anhydride, acetyl chloride and acetyl-imidazole. Furthermore, ithas been found than when used in the presence of aqueous solutions suchas blood, acetic anhydride is less liable to hydrolysis and thereforeinactivation than reagents such as methoxyacetic anhydride orchloroacetic anhydride. Therefore it is necessary to use acylatingreagents that are not so unstable in aqueous solutions that theyhydrolyse before they can modify the nucleic acid, whilst modifying thenucleic acid with chemical groups that can be removed without leading tonucleic acid destruction. This is particularly problematic for RNA whichis readily degraded by the types of alkalis that are efficient atremoving the acyl groups. Methods of using such alkalis for thedeprotection of the 2′-OH groups of RNA have been described elsewhere(WO/01/94626, WO/00/75302).

Numerous methods have been described to remove acetyl groups fromacylated compounds including enzymatic, electrolytic and chemical means.Whilst enzymes such as esterases and lipases have found widespread usedue to their mild reaction conditions, they are expensive, are oftencontaminated with non desired proteins or compounds and require carefulpre-selection to find an appropriate activity. They are also sensitiveto the charge on the acylated compound so that strongly chargedmolecules such as nucleic acids may not be good substrates. However, theuse of suitable enzymes in particular esterases and lipases fordeprotection of modified nucleic acids would be extremely useful.

Whilst many chemical deprotecting methods are also known for removingacetyl groups (Protecting Groups in Organic Synthesis, Greene and Wuts,2^(nd) Edition, Wiley Interscience) most if not all involve either abase or acid, conditions that are likely to lead to extensive cleavageof the desired RNA during deprotection.

Methods for protecting RNA by chemical modification have beenextensively described in Patent application numbers WO/01/94626 andWO/00/75302. Fully or even partially acetylated modified RNA isprotected from degradation from nucleases, however it is not capable ofserving as a reverse transcription template and neither cannot ithybridise. Modified RNA can be stored, transported and archived in itsprotected form at ambient temperature instead of, as, is more usual inthe industry, on ice, dry ice or in liquid nitrogen. Prior to analysisthe modifications are removed. It is therefore important, after the RNAhas been protected to be able to remove the acetyl groups in order toallow efficient reverse transcription, hybridisation and to serve as atemplate for protein synthesis. Methods to remove acetyl groups from RNAhave been described in patent application numbers WO/01/94626,WO/00/75302. These methods include the use of potassium cyanide, HunigsBase, dimethylethylenediamine, sodium hydroxide and ammonium hydroxide.

Unfortunately, RNA is extremely sensitive to the presence of alkali andRNA chain cleavage occurs after the acetyl group has been removed fromthe 2′-OH position. It is therefore necessary to use a pre-calibratedamount of alkali sufficient to remove the acetyl groups from the RNA butnot so much that it leads to significant subsequent RNA cleavage. Whilstthis has been achieved by the use of mixtures of either sodium hydroxideor ammonium hydroxide with alcohol, some RNA chain cleavage is aninevitable result of acetyl deprotection using these methods (disclosedin patent applications; WO/01/94626, WO/00/75302). We have tested manyother methods and chemicals have been tested for their activity toremove acetyl groups from RNA without leading to its cleavage includingsodium hydrogen carbonate, sodium carbonate, potassium carbonate,potasium hydroxide, triethylamine, guanidine, hydrazine and HCl. Thesecompounds either had no activity or lead to some degree of RNA chaincleavage and are therefore not the preferred method for deprotection ofRNA although they are useful for the removal of acyl groups from DNAoligonucleotides and polynucleotides when the acyl group is eitherattached to the 5′-OH, the 3′-OH or the nucleobases. DNA is much lesssensitive than RNA to being cleaved by alkalis. For example DNA can beincubated in 1M NaOH for one hour with no detectable degradation,conditions which would reduce RNA to monomers within 5 minutes. Othersmethods for the removal of protecting groups from the nucleobase ofoligodeoxynucleotides but not the 2′-position of RNA polymers, that havebeen described in the literature as either mild or ‘ultra-mild’deprotection methods (Glen Research, USA), are completely inappropriatefor RNA because they would be expected to lead to extensive chaindegradation. Furthermore, both methylamine and ammonium hydroxide whichare widely used for are toxic and dangerous to work with and very strongsmelling. They are not therefore suited to daily laboratory settings andmust be used in a fume hood.

One published method (Boal et al., Nucleic Acids Res. (1996)15:3115-3117) describes a method to deprotect benzoyl, isobutyrl orisopropoxyacetyl nucleobases on oligonucleotides following synthesis,employing gaseous ammonia at 10 bar pressure or methylamine at 2.5 barin a pressured device. However, no mention is given of the utility ofthis method for removing modifications from the 2′-position of nucleicacids, rather the gaseous amines are used only for deprotecting thenucleobases of oligodeoxynucleotides. The method therefore does notmention deprotecting polynucleotides, RNA or the 2′-OH position all ofwhich are useful to practice the invention as we have set out here. Itis not expected that pressurised use of gases will find wide spreadacceptance in laboratories. We have unexpectedly demonstrated thatprimary amines can not only remove protecting groups from the 2′-OHposition of RNA but also lead to only limited cleavage of thephosphodiester backbone of RNA as would be expected for a base. It wasalso unexpected that primary amines such as ethylenediamine andtriethylenetetramine reduce the amount of protein contamination bindingto metal ions such as those present in hydroxylapatite.

It has been found that either modified RNA or RNA that has beenimmobilised on a solid phase such as charged nylon (Hybond N+, AmershamBiosciences, UK) and then treated with an alkali such as sodiumhydroxide or ammonium hydroxide is more protected from subsequent alkalidegradation than similar RNA in solution. This may be because theimmobilised modified RNA or RNA has limited torsional freedom, that isthe 2′-hydroxyl group in the presence of alkali and a solid phase cannotsubsequently attack and cleave the 3′ phosphodiester bond as would occurin solution. The cleavage effect of the alkali on the RNA is thereforereduced. Whilst this method is useful for deprotecting modified RNA forhybridisation purposes such as northern blotting, it has limitedapplication for deprotecting modified RNA for reverse transcription ortranscription mediated amplification (TMA) because polymerases such asreverse transcriptases cannot effectively copy immobilised RNA. Methodsfor deprotecting modified RNA on solid phases have been set out inPatent application numbers (WO/01/94626, WO/00/75302).

Other solid phases that provide only a temporary reversible binding ofthe modified RNA to the surface such as silica (Qiaex II particles(Qiagen, Germany) were found not to be useful for deprotection of RNAwith aqueous alkalis because the RNA is eluted from the surface into thealkali during deprotection leading to its cleavage at normal rates.Other solid phases such as hydroxylapatite, although they bind RNA evenin the presence of alkali, did not provide the level of protection fromcleavage provided by charged nylon.

However, when acetyl modified RNA is bound onto a solid phase such assilica or hydroxylapatite it can be effectively deprotected withoutsignificant cleavage using dry gaseous ammonia. In this example, thesolid phase may not be reducing the amount of cleavage, rather it maysimply present the modified RNA to the ammonia such that a substantialproportion of the acetyl groups are readily accesible. By contrast, aprecipitated or spin-dried pellet of RNA is less readily deprotected byammonia gas possibly because of the difficulty of the ammonia enteringthe pellet and contacting all the acetyl groups. It is important thatthe ammonia vapour is dry because it has been found that the presence ofwater leads to RNA cleavage.

Pressurised bottled ammonia is a suitable dry source or alternatively,ammonia can be conveniently distilled from a solution of 28% ammoniumhydroxide. In the latter example, it is important to pass the ammoniavapour over at least one surface to condense any water vapour that mayhave been carried over with the ammonia from the ammonium hydroxidesolution. The dried ammonia can then be passed into a flask containingone or more tubes bearing the modified RNA bound onto beads.Deprotection times vary from a few minutes to one hour, depending on (i)the concentration of ammonia, (ii) the temperature, and (iii) the amountof RNA exposed to the ammonia in the particle pellet. It has been foundthat dense pellets of silica or hydroxylapatite bearing the modified RNAare deprotected more slowly than diffuse pellets of particles.Controlling the pellet size can be difficult and therefore it can bedifficult to judge the necessary deprotection time. Deprotected RNA canbe simply washed to remove traces of the by product ammonium acetate andeluted for downstream applications. Unfortunately this method issomewhat cumbersome and unpredictable for routine use and involvesworking in a high performance chemical fume hood.

An improved method of deprotection has been developed that does notinvolve-gaseous materials. Although the use of methylamine (CH3-NH2) isknown for the removal of acyl groups from nucleobases ofoligonucleotides, it is a dangerous, gaseous, toxic chemical to usewhich would have unkown effects on fragile molecules such as polymers inparticular RNA polynucleotides. One of the problems is that methylamineis its low boiling point (−6° C.) and a high vapour pressure (2250 mm Hgat 20° C.) so that evaporation rates are very high indeed. Similarlyammonia boils at −33° C. and has a high vapour pressure making its usedangerous. Both ammonia and methylamine are gases at room temperatureand pressure. This preferred method involves the use of primary aminecompounds with vapour pressure below 2000 mm Hg, more preferably below1000 mmHg, even more preferably below 500 mm Hg and most preferablybelow 200 mm Hg at 20° C. This preferred method involves the use ofprimary amine compounds with boiling points above 0° C., more preferablyabove 25° C., even more preferably above 50° C. and most preferablyabove 100° C. at 1 atmosphere of pressure.

Long chain (containing five carbon atoms or more) primary aminecompounds such as pentylamine have boiling points above 100° C.,however, the rate of deprotection with these compounds is expected to beslower than for shorter (less than five carbon atoms) chain primaryamines such as butylamine because of steric hinderance between thenucleic acid and the bulky long chain amine. There is therefore atrade-off between the rate of deprotection of the primary amine and it'sboiling point/volatility. However, advantageously, compounds that canextensively hydrogen bond such as ethanolamine are not only relativelysmall molecules thereby reducing steric hinderance but they also haveboiling points above 100° C. because of hydrogen bonding. Thereforeprimary amine containing compounds that can also hydrogen bondextensively are both useful because they deprotect quickly but alsobecause they have significantly lower vapour pressures so they are easyto handle. Molecules with two or more amine groups such asethylenediamine are preferred to monamines such as ethyleneamine becauseof their greater reactivity, higher boiling and flash points and lowervapour pressures.

Examples of suitable primary amine compounds for deprotection includeethanolamine (Fluka, USA), ethylenediamine (Fluka, USA),diethylenetriamine (Fluka, USA), triethylenetetramine (Fluka, USA),tetraethylenepentamine (Fluka, USA). Other primary amines includediglycolamine agent (Huntsman, USA), Jeffamine (polyoxyalkyleneamine)type molecules including Jeffamine ED which is water soluble (Huntsman,USA), dimethylaminopropylamine (Huntsman, USA) and methoxypropylamine(Huntsman, USA).

It has also been found that other primary amine containing compoundssuch as the amino acids Lysine and Arginine are good reagents for thedeprotection of acylated nucleic acids, in particular acylated RNA.These amino acids contain both the α-NH2 and the ε-NH2 groups, both ofwhich, in the free base form, may contribute to the removal of acylgroups. Advantageously, these amino acids compounds have a very lowvolatility, are relatively cheap, widely available and create fewdisposal problems as they are biodegradable. Indeed they are excellent‘green chemistry’ solutions. Lysine and arginine can be purchased eitherin their free base form or as their salts, the free base forms arepreferred

Such deprotection reagents as ethanolamine, ethylenediamine,diethylenetriamine, triethylenetetramine, lysine and arginine may alsobe useful for removal of unwanted modifications of RNA and DNA madeduring the use of diethyl pyrocarbonate (DEPC), a widely used inhibitorof RNases. The non desired DEPC modifications of nucleic acids forexample on the nucleobases, lead to reduced nucleic acid templateefficiency, therefore their removal is desired if fall nucleic acidtemplate activity is to be restored.

Advantageously it has been found that deprotection can be carried outwhilst the RNA is immobilised on a solid support, particle or bead orother solid phase such as hydroxylapatite or silica. The solid phase mayconsist of an organic or inorganic particle, a polymeric linear,globular or cross-linked molecule or resin. It may be made of a varietyof materials or material composites such as acrylamide, agarose,cellulose, polyamide, polycarbonate, polystyrene, polyethylene,polypropylene polytetrafluoroethylene, nitrocellulose, latex, aluminium,copper, nickel, iron, a metal oxide, a mixture of metals such as aniron-zinc blend or an oxide thereof, lithium iron III oxide, glass,hydroxylapatite or silicon.

The solid phase may also be a composite between a particle or surfaceand an immobilised nucleic acid, in particular an oligonucloetidecomplementary to the desired target analyte nucleic acid. Preferably thesolid phase bearing the complementary oligonucleotide is a magneticparticle thereby aiding handling during purification. In order to alloweffective capture of an analyte RNA using a complementaryoligonucleotide, the RNA must be at least partially deprotected prior tohybridisation, because acetylation of RNA at the 2′-OH position reducesor abolishes the capacity of the RNA to hydrogen bond with acomplementary oligonucleotide. It is particularly convenient to protectthe analyte RNA molecule and then to deprotect and capture it with acomplementary oligonucleotide in the presence of a primary aminedeprotection reagent.

The solid phase may also possess specific properties that aid in themanipulation of the particle such as paramagnetic or magneticproperties, a diameter allowing retention by a filter, an increaseddensity that enhances sedimentation or separation by centrifugation orincorporate a tag aiding identification, capture or quantification ofthe particle. This solid phase provides a simple means to separate thedeprotection reagent from the nucleic acid sample after deprotection.However, primary mines such as ethanolamine, ethylenediamine,diethylenetriamine, triethylenetetramine, lysine and arginine can beused to deprotect the RNA when the RNA is either (i) attached to a solidsupport (see example 23), or (ii) in solution (see example 1).

Primary amines for use in this invention include; C1-C10 alkylamines,C1-C10 aminoalkylamines, C2-C10 hydroxyalkylamines, C2-C10haloalkylamines, C4-C10 di(aminoalkyl)amines, C4-C20dialkylaminoalkylamines, C4-C20 alkyloxyalkylamines and C3-C10diaminocarboxylic acids

Very usefully, it has been found that primary amines such asethanolamine, ethylenediamine, diethylenetriamine, triethylenetetramine,lysine and arginine not only serve as useful deprotection reagents butthey also reduce the amount of non-desired proteins that bind tohydroxylapatite and silica during nucleic acid purification. Addition of200 μl of ethylenediamine to a 1.45 ml reaction containing 200 μl ofplasma, 50 μl of 1-methylimidazole and 1.2 ml of tetrahydrofuran/aceticanhydride (2:1 vol:vol) has been found to reduce protein binding by 7.5fold whilst not affecting RNA yield. This is an unexpected resultbecause hydroxylapatite and silica surfaces are well known to bindproteins. This highly desirable property of primary amines and to alesser extent non-primary amines such as triethylamine, pyridine, TEMED,dimethylpropylamine and dimethylethylenediamine can be used to reducethe amount of proteins binding to the solid phase during purification ofRNA and DNA from a biological sample. It is known that ethylenediamineand triethylenetetramine can bind to metal ions by coordination and itis perhaps this ‘chelating’ to calcium on the hydroxylapatite thatpartly explains the reduced protein binding. It is preferable, but notessential to add the amine premixed with the hydroxylapatite prior toaddition to the sample, but no advantage was noted by incubating thereaction with ethylenediamine for periods longer than 10 min. prior toadding the hydroxylapatite.

Hydroxylapatite binds to charged biomolecules by a combination of itspositively charged calcium ions, negatively charged phosphate ions andhydroxyl groups. Although nucleic acids such as RNA and DNA carry astrong negative charge allowing binding to the hydroxylapatite calciumions, so also do many proteins. Furthermore, many proteins are alsopositvely charged so that they bind to the phosphate groups of thehydroxylapatite. Frequently, the nucleic acid is in very lowconcentration compared with the contaminating proteins, lipids andcarbohydrates present in the cell or biological sample such as blood,serum etc. For example, blood contains approximately 60 000 times (w:w)more protein than RNA. As a result it can be difficult to purify thenucleic acid away from the contaminants using hydroxylapatite. Mostmethods have relied on using differential elution to separate thenucleic acids from the contaminants, however, such preparations areeither low in the desired nucleic acid or contain significant amounts ofthe non desired contaminant. The contaminant may reduce the efficiencyof downstream applications of the nucleic acid such as reversetranscription, PCR, hybridisation or even lead to its degradation ifsignificant amounts of nucleases are eluted with the nucleic acidanalyte.

Therefore, inhibiting proteins from binding to the hydroxylapatitewhilst still allowing deprotection and nucleic acid binding to occur ishighly advantageous. We have found that purification of nucleic acids,in particular RNA from serum and plasma has been found to be improved bythe addition of the deprotection amine reagent to the chemicalmodification reaction bearing the acylated RNA. For the purification ofmodified RNA from biological samples such as blood plasma withoutdeprotection occuring but still inhibiting proteins from binding thepurification matrix such as silica or hydroxylapatite can beaccomplished by using secondary and tertiary amines. Secondary andtertiary amines have been found to inhibit proteins from binding tosilica and hydroxylapatite but do not lead to deprotection of the RNA.Therefore, in this example, modified RNA can be purified from complexbiological samples by the addition of a secondary or tertiary amine tothe reaction before, or at the same time as the purification matrix isadded to the mixture. The modified RNA can then be purified and elutedfrom the matrix in its protected form. The modified RNA can also bestored and archived in its protected form and deprotected using aprimary amine immediately prior to use for example hybridisation orRT-PCR.

Likewise, DNA can be separated from contaminating proteins by the use ofprimary, secondary and tertiary amines in conjunction with the solidphase matrix such as silica or hydroxylapatite beads. The purpose of theamine in this case is not only to remove any potential acetyl groupsattached to the DNA, for example at the 5′ and 3′ OH positions but alsoto inhibit the binding of proteins to the purification matrix.

Modified RNA can be deprotected at any one of three points duringpurification (i) at the same time as binding to the solid phasepurification matrix as described in example 1, (ii) after the modifiedRNA has been been bound to the solid phase purification matrix, butbefore elution, and (iii) after elution from the solid phasepurification matrix. The advantage of (i) is that the modified RNA isdeprotected and proteins removed at the same time, the advantage of (ii)is that the deprotected RNA can be readily removed from the deprotectionreagent by means of the solid phase it is bound to, whilst the advantageof (iii) is that the RNA is purified in its modified and thereforeprotected form. However, with method (iii) there is a necessity toseparate the deprotected RNA from the deprotection reagent. This can beachieved by filtration using a Centricon device (Millipore, USA)according to maufacturer's instructions, precipitation using alcohol ordialysis. Alternatively, the RNA can be separated by evaporating thedeprotection reagent away using reduced pressure or increasedtemperature. In this case, a deprotection reagent with a decreasedboiling point is preferable to one with a high boiling point. Primaryamine reagents with a reduced boiling point are propylamine (bp 47° C.)or butylamine (bp 76° C.). Alternatively, the deprotected RNA can beremoved from the deprotection reagent by binding it onto a solid phasedeprotection reagent such as hydroxylapatite, washing the beads uding70% ethanol to remove excess deprotection reagent, and then eluting theRNA using phosphate or a chelator.

The amine may also be immobilised on a solid phase so that convenientlythe deprotection reagent on for example a bead, can be mixed with theprotected RNA, deprotection allowed to proceed and then it can beremoved from the deprotected RNA on the basis of a property of the solidphase. This property can be a paramagnetic core or its collection duringcentrifugation or filtration. Suitable materials include ethylenediaminepolymer bound (Aldrich, USA catalogue number 47,209-3)

The use of magnetic or paramagnetic preparations of hydroxylapatite oreven silica is preferred because of the ease of handling, washing andthe large surface area of particles compared with other forms of thesolid phase such as a membrane.

Solvents

An alternative solvent to tetrahydrofuran for the modification ofnucleic acids, in particular RNA, is dioxolane, a water miscible solventwith a lower vapour pressure and higher boiling point thantetrahydrofuran. However, dioxolane like tetrahydrofuran, producespotentially dangerous flammable vapours. Other solvents with much lowervapour pressures and much higher flash points that have been found to beexcellent alternatives to either dioxolane or tetrahydrofuran, includeN-methylpyrrolidinone (NMP), Formyl morpholine, Dimethyl imidazolidone,acetoxy acetone and acetonyl acetone. Solvents are preferred with flashpoints above 37° C., more preferably above 60° C. and even morepreferably above 90° C. Particularly preferred areN-methylpyrrolidinone, Formyl morpholine, Dimethyl imidazolidone, theirphyscial properties compared with tetrahydrofuran are set out inTable 1. Surprisingly, these three solvents have a higher capacity todissolve complex biological samples such as blood and plasma proteinsthan does either tetrahydrofuran or dioxolane so that less precipitatesform during handling of mixtures of biological samples and organicmixtures, thereby increasing nucleic acid yield and reducing proteincontamination of the nucleic acid. For example 200 μl of blood can bedissolved to form a homogenous mixture in 1 ml of N-methyl pyrrolidinonewhilst the same amount of blood mixed with either tetrahydrofuran ordioxolane causes the proteins to rapidly precipitate out of solution.N-methylpyrrolidinone, Formyl morpholine and Dimethyl imidazolidone mayfind other uses in life sciences for dissolving for example recombinanttherapeutic proteins that are insoluble when released using traditionalmethods from cells or cell free translation systems. Bothtetrahydrofuran and dioxolane require the presence of approximately 5%of 1-methylimidazole in order to dissolve up to 20% final volume ofhuman plasma, whilst N-methylpyrrolidinone, Formyl morpholine, Dimethylimidazolidone can completely solubilise this amount of plasma withoutany addition of 1-methylimidazole. Advantageously, N-methylpyrrolidinoneis biodegradable improving disposal issues. TABLE 1 Comparison of theproperties of various solvents Water Solvent Flash point solubilityOther properties Tetrahydrofuran −17° C. complete — Dioxolane  −3° C.complete — N-methyl pyrrolidinone  91° C. complete biodegradable Formylmorpholine 125° C. complete Compatible with polycarbonate Dimethylimidazolidone 102° C. completeReactant

It has been found that acrylic anhydride, propionic anhydride or butryicanhydride (Fluka Chemicals, USA) can replace acetic anhydride inexample 1. The advantages of these reagents is that they may have longershelf storage lifetimes compared with acetic anhydride and they may alsobe more stable when premixed with the solvent and catalyst as set outbelow. These reactants have been described in (WO/01/94626 andWO/00/75362).

The reactant, catalyst and solvent can be predispensed in a bloodcollection tube so that the blood sample is drawn directly from thepatient or blood collection pouch into the tube where it contacts thereactant and the nucleic acids are stabilised. The blood collection tubecontaining the stabilised nucleic acids can then be transported,archived or used directly to extract and purify the nucleic acids. Theadvantage of this approach is that a single tube contains all thenecessary reagents necessary for nucleic acid stabilisation, therebyminimising the manual steps required to initiate the stabilisationreaction. In one manifestation of the device, blood is drawn directlyfrom the vein of the patient, via a needle and tube into theappropriately marked tube containing both the necessary reactants and avacuum sufficient to induce blood flow into the tube. In another format,blood is drawn from the patient using a syringe and needle and theninjected by way of the needle and a rubber septum at the top of thetube, into the reactant. Complete mixing is assured by gentle inversionof the tube. The tube and septum or top of the tube are preferablycomposed of materials that are chemically compatible with the reagents,such as those composed of polypropylene, polyethylene, glass or PTFE.Such a device is particularly suited for diagnostic purposes for exampleof, viruses such as HIV and HCV.

Catalysts

The advantage of 1-methylimidazole as an acylation catalyst is that italso serves as a very effective buffer so that the acidification of thereaction caused by the accumulation of acetic anhydride is buffered.Additionally, 1-methylimidazole is an excellent solvent for dissolvingbiomolecules such as proteins. However, surprisingly, other catalystshave been successfully used including 4-pyrrolidinopyridine and2-hydroxypyridine as set out in example 31. However, it has been foundthat whilst a mixture of either 1-methylimidazole, N-methylpyrrolidinone and acetic anhydride or 4-pyrrolidinopyridine,N-methylpyrrolidinone and acetic anhydride retains its acylatingactivity after storage for 11 days at ambient temperature, the mixture2-hydroxypyridine, N-methylpyrrolidinone and acetic anhydride loses itsactivity after five days. Further comparison are set out in example 30.Therefore the use of the catalysts 4-pyrrolidinopyridine and1-methylimidazole is preferred for storage of mixtures containingacylating reagents. These catalysts have been described in (WO/01/94626and WO/00/75302).

Volumes of Reagents

For the purification of nucleic acids from a 200 μl plasma or serumsample according to example 1, 1.25 ml of organic reagents (catalyst,solvent and reactant) are added to the sample. It has been found thatonly 1.05 ml of organic reagents are necessary for the modification,stabilisation and purification of the RNA. The reduced reaction volumesalso means that less deprotection reagent volumes can be added, thevolumes of the deprotection reagents can be reduced from 3.4 ml to 2.6ml as set out in example 27 below.

It has surprisingly been found that the nucleic acid analyte can bebound onto the hydroxylapatite beads prior to the application of thedeprotection agent as set out in example 28, The advantage of thisapproach is that a smaller volume of deprotection reagent can be addedto the hydroxylapatite/RNA complex because it does not become quenchedby the acetylating reagent present in the reaction. In example 1, arelatively large amount of both 1-methylimidazole and ethylenediamineare added at the same time as the hydroxylapatite beads because theacetic anhydride/acetic acid present in the reaction reacts with thesecomponents. Using the method as set out in example 28, thehydroxylapatite beads are first added to the reaction, and followingbinding of the RNA to the beads the reaction containing the aceticanhydride/acetic acid can be removed, the beads washed and then a smallamount of deprotection reagent added. It has been found that the volumeof the deprotection reagent required is reduced from 3.4 ml to 0.2 ml orless.

It has been found that addition of long of carrier RNA in the reactionimproves the RNA yield 1.3 fold. This is probably due to suppressingnon-specific interactions between the RNA anlayte and plasticware. Bothpoly rC (Midland Certified Reagent Company. USA) and total yeast RNA hadsimilar effects. Carrier RNA also improved the recuperation of theanalyte RNA following ultracentrifugation using a Microcon-YM-50 deviceby 1.25 fold.

Hydroxylapatite Beads

Hydroxylapatite magnetic beads are commercially available (Chemicell,Germany), however, due in part to the organic reagents and in part tothe density of the liquids with which the beads are mixed, complete beadcollection using a fixed magnetic stand (Promega, USA) is less thancomplete. It has been noted that a proportion of the HXA Type I beadpopulation is composed of much smaller particles, that nevertheless binda significant proportion of the desired nucleic acid in the sample. Theloss of these smaller particles represents an important reduction in theoverall yield of nucleic acid. Complete collection of all particles canonly be achieved by centrifugation of the solution containing theparticles at 14 000×g for 5 minutes or filtering the mixture using anAcrodisc GHP (13 mm) and a syringe (Catalogue number PALL 4556, PallInc, USA). It has been found that by using this Acrodisc filter that theoverall RNA yield increases from 80 to 100%. In order to avoid the useof filters, hydroxylapatite beads were selected with an increasedcontent of larger particles that were more eaily collected using amagnet as follows. 1 ml of hydroxylapatite particles was added to 50 mlof 80% glycerol, mixed, then centrifuged at 3000×g for 10 minutes. Thesupernatant containing the smaller particles was discarded and thepellet washed in 20 ml of water and resuspended in 1 ml of water.Hydroxylapatite beads prepared in this way are more effectivelycollected by magnetic collection than the non-size selected beads. Ingeneral, magnetic-hydroxylapatite beads are prepared that (i)efficiently bind nucleic acids with relatively little proteincontamination, (ii) can be collected easily using a fixed magnet, (iii)do not disintegrate or dissolve in the presence of organic or aqueoussolvents or reactants and (iv) readily release the nucleic acid onapplication of the elution solution and (v) allow the differentialrelease of RNA, DNA and proteins in distinct fractions for subsequentanalysis of each fraction.

It has been found that for magnetic-hydroxylapatite bead type 14/2(Chemicell, Germany), the optimum amount of beads for RNA purificationis 25 μl (50 mg/ml). Addition of more beads had the effect of reducingRNA yield whilst increasing significantly the protein contamination.

Pretreatment of Magnetic Hydroxylapatite

It has been found that pretreating hydroxylapatite beads (Chemicell,Germany) with 0.2M sodium phosphate (Sambrook et al., (1989) MolecularCloning: A Laboratory Manual, CSH.) before use, as set out in example 1,improves the ratio of RNA to protein bound to the beads. This isprobably because the sodium phosphate reduces the overall charge on thesurface of the beads thereby favouring the binding of more highlycharged molecules such as nucleic acids over less charged such asproteins thereby increasing the yield and purity of the desired nucleicacid. Whilst sodium phosphate is effective in this function, organicphosphates such as 0.2M glucose phosphate, cellulose phosphate or serinephosphate (Sigma-Aldrich, USA) did not have the desired effect ofincreasing nucleic acid yield and purity, rather, RNA yield wasdecreased compared with sodium phosphate treated hydroxylapatite.However, protein contamination was also reduced suggesting that it maybe possible to pre-treat the beads with a phosphate that has the desiredproperty of reducing protein contamination whilst increasing RNA yield.It is expected that other soluble phosphates such as potassium phosphatewould have a similar desirable effect to sodium phosphate. Examples ofpotentially useful compounds include D-arabinose-5-phosphate,D-6-Fructose-phosphate, D-glucosamine-6-phosphate,D-mannose-6-phosphate, D-ribose-5-phosphate, D-ribulose-5-phosphate,D-glyceraldehyde-3-phosphate, D-sorbitol-6-phosphate, DL-ascorbicacid-2-phosphate, glycerol phosphate, ammonium phosphate, bariumphosphate, bismuth III phosphate, boron phosphate, citrate phosphate,cobalt phosphate, copper II phosphate, threonine phosphate, nucleotidemonophosphate, nucleotide diphosphate and nucleotide triphosphate.

Types of Nucleic Acid Binding Solid Phases

Although hydroxylapatite is efficient at binding nucleic acids from anorganic-aqueous mixture, other types of solid phases can also be used.These include silica such as Qiaex II (Qiagen, Germany), derivitisedsilica such as Magnisil (Promega, USA) and surprisingly simply iron IIIoxides (<5 μm particle size) (Aldrich, USA, catalogue number 31,006-9).Nucleic acids can be removed from iron oxides by mixing the particleswith 0.1M sodium phosphate buffer (pH 6.8) or alternatively using 100 mMEGTA-TEAH salt (pH 102). It has been found that Iron III chloride, 5 wt% on silica gel (Aldrich, USA, catalogue number 36,100-3) and largepieces of Iron III oxide (>2 mm diameter pieces) are both very effectiveat binding nucleic acids from both aqueous and organic reactions.Elution can be brought about by the addition of 0.2M Sodium phosphate or10 mM EGTA, pH 10.2.

Elution and Removal of Chelator

In the preferred method of practising nucleic acid purification, as setout for example, below, nucleic acids are eluted frommagnetic-hydroxylapatite using a chelator such as EGTA, EDTA, DTPA,HEDTA, NTA and BAPTA. However, the presence of the chelator with thedesired nucleic acid can inhibit the activity of enzymes requiringdivalent metal cations such as Tth DNA polymerase. These enzymes arenecessary for downstream analysis of the nucleic acid. Therefore thechelating activity is desired for elution but undesired followingelution. The chelating activity present with the nucleic acid solutiontherefore needs to be removed. A minimum amount of the chelator shouldbe used that is necessary for elution of the desired analyte so thatonly a minimum is present with the eluted nucleic acid.

It has suprisingly been found that the type of salt of the chelatingagent is extremely important for the chelating activity and hence thecapacity to release the nucleic acid from the magnetic-hydroxylapatite.For the salts tested, sodium and potassium salts of EGTA were the leasteffective whilst, ammonium salts, prepared using ammonium hydroxideaddition to the free acid form of EGTA, was significantly better. Themost effective salts of EGTA were found to be tetramethylammonium,tetraethylammonium and tetrabutylammonium at pH 9.9 all of which wereapproximately equivalent. These salts were approximately 1.9 fold moreeffective than the ammonium salt of EGTA at releasing RNA frommagnetic-hydroxylapatite. Tetraalkylammonium salts of chelators such asfor example, EDTA, NTA, BAPTA and EGTA may be useful for otherapplications requiring strong chelators.

The pH of the elution solution also has a profound effect on itscapacity to release the nucleic acid from magnetic-hydroxylapatite.Whilst for example, a solution of 10 mM EGTA pH 8 was poor at releasinga 32P labelled RNA sample from magnetic-hydroxylapatite, there was animprovement, compared with 10 mM EGTA (pH 9), of 1.12 fold (pH 9.3), 1.4(pH 9.6), 1.5 (pH 9.9) and 1.45 (pH 10.2). The optimum pH was thereforedetermined to be approximately pH 9.9. The use of chelating solutionsthat have maximum activity is critical for reducing the elution volumeto a minimum. For example, using a solution of 10 mM EGTA pH 8.7 (sodiumsalt), the minimum volume for complete RNA elution was found to be 400μl, whilst for a solution of 10 mM EGTA pH 8.7 (tetraethylammonium salt)the volume could be reduced to 50 μl.

One method for chelator removal is ultracentrifugation using filterunits with MWCO of 5 000 to 100 000 daltons. Processing of samplestherefore requires centrifugation as set out in example 1 and issignificantly faster than dialysing solutions to remove the chelator.However there are alternative methods for removing the chelator from theRNA as set out below.

Methods for removing the chelating activity include usingphoto-sensitive chelating reagents (so called ‘caged calcium’ reagents)such as NITR-5, NITR-7 (U.S. Pat. Nos. 4,689,432 and 4,806,604),nitrophenyl-BAPTA (Graham et al., (1994) Proc. Natl. Acad. Sci. 91:187),NP-EGTA and DMNP-EDTA. These reagents are photo-sensitive and destructon exposure to short bursts of light of the correct wavelength.Therefore the non-desired chelating activity can be destroyed bysubjecting the eluted nucleic acid/chelator solution to light of theappropriate wavelength and exposure. For example, NP-EGTA has a 12 000fold reduction in its affinity for calcium on exposure to uv light.Methods, materials and references describing the use of some of thesereagents for the release of intra-cellular metal ions have beendescribed (Handbook of Fluorescent Probes and Research Chemicals,Chapter 20, Molecular Probes). Nucleic acid-photosensitive chelatingmixtures could be exposed individually or en masse to the light sourcewhilst present in microcentrifuge tubes or 96 well plates. Thephoto-destruction of the chelator should be achived with the smallestamount of light necessary to achieve sufficient cleavage so that minimumphoto-destruction occurs to the nucleic acid analyte.

An alternative use of photosensitive chelators is as follows. Manyimportant enzymatic reactions such as PCR and reverse transcriptionrequire divalent metal cations, in particular Mg and Mn for activity,however premature activity of these enzymes particularly at a reducedtemperature can lead to non-specific polymerisation products. Severalmethods have been employed to reduce premature polymerisation occurungincluding so-called ‘hot-start’ methods where the enzyme is added to thereaction only after the apprpriate temperature has been reached, or theenzyme only becomes active at the correct temperature. However these areeither likely to increase contamination or are expensive. Alternatively,a photo-sensitive chelator could be added to the reaction at aconcentration equal to the amount necessary to remove all the divalentmetal cations present, thereby inhibiting the enzyme activity. Once thecorrect reaction temperature has been obtained, the reaction can beexposed to an appropriate light source thereby destroying the chelatorand releasing the divalent metal cation and initiating thepolymerisation reaction. The light source could be for example onealready located in the amplification machine (LightCycler, Roche, USA)or an external source. Another method for removing chelating activity isto use an antibody specific for the chelating agent, for exampleanti-BAPTA IgG (Cell Calcium 21:175 (1997); Cell Calcium 22:111 (1997)which is commercially available (Molecular probes Inc, USA). In thisexample the elution solution would be BAPTA, and the anti-BAPTA antibodywould be tethered directly or via secondary antibody to a solid phasesuch as protein A-sepharose (Sigma-Aldrich, USA) and the elutionsulution incubated with the antibody allowing the chelator but not thenucleic acid analyte to be removed.

Yet another method to remove the chelating activity would be to use a‘tagged’ chelator. The tag could be for example, a biotin moleculeattached to the chelator DTPA. Following elution, the chelator isremoved from the nucleic acid using streptavidin or an avidin solidphase that are commercially availbale from a number of vendors (e.g.Dynabeads M-280, Dynal, Norway). Methods for attaching the chelator DTPAto other molecules has been described (Hnatowich (1983), Science220:613) and materials are commercially available such as EDTAmaleimdo-C5-benzyl (Calbiochem Inc, USA) or biotin-chelator iscommercially available as biotin-DTPA (catalogue number D1534,Sigma-Aldrich, USA).

Magnetic Collection

We have found that using an external magnet such as those commerciallyavailable (Promega, USA, Dynal, Norway) offer an effective method tocollect large magnetic or paramagnetic beads from solution. However,when the liquid containing the magnetic beads is viscous and/or a volumegreater than 5 ml, the rate of bead collection is reduced leading tolosses. An alternative to an external magnet is the use of a disposablemagnet that is added directly into the tube containing the magneticbeads. The distance between the magnet and beads is therefore markedlyreduced thereby increasing the magnetic force exerted on the magneticbeads and improving bead collection. Suitable types of disposablemagnets are those that have a non-reactive non-contaminating surfacesuch as PTFE. These are commercially available as magnetic stirring barsused to dissolve solids into liquids. Examples of products includeAldrich catalogue numbers; Z42,022-0, Z32,866-9 and Z32,868-5 all ofwhich are relatively cheap devices. The shape of the magnetic stirrer isnot particularly limited, but those with sufficient surface area to bindall the beads in the liquid without the beads overlapping one anotherare preferred. It has been found that a 2 cm long, 6 mm diameter stirreroffers more than sufficient surface area to effectively bind 2.5 mgmagnetic hydroxylapatite Type I (Chemicell, Germany). Usefully, themagnetic stirrer after it has been added to the liquid containing thebeads can be manipulated in a sealed tube by the use of an externalmagnet, so for example, it can be lifted out of the liquid, allowing theliquid to be removed and the stirrer washed by the addition of freshreagents. Surprisingly, the magnetic beads can be temporarily removedfrom the stirrer by gently vortexing the tube containing the stirrer,beads and a liquid, allowing efficient washing of the beads to occur.The stirrer could have a central hole allowing a disposable plasticpipette tip to insert through it and remove liquid around and beneaththe stirrer thereby improving the dispensing and removal of for example,wash solutions. Low protein binding surfaces such as PTFE orpolypropylene are preferred because they reduce protein contaminationand are compatible with solvents and reactants such as tetrahydrofuranand acetic anhydride.

It has also been found that magnetic-hydroxylapatite beads can bequickly collected by placing two electrodes in the solution containingthe beads and connecting the leads to a 9 volt battery. Themagnetic-hydroxylapatite beads collected on both the anode and cathodecompletely coating the surfaces. The beads could then be released fromthe electrodes for washing or elution by turning off the current. Theefficiency of the collection be be improved by the addition of anelectrolyte such as 0.5×TAE electrophoresis buffer.

Magnetic Mixing

It has been found that specialised magnetic mixers such as the MCB1200(Dexter Magnetic, UK) are effective for agitating solutions containingmagnetic beads. However, it is relatively expensive device and islimited to 12 available tube spaces. Surprisingly it has been found thatmagnetic stirrers designed for use with magnetic stirring bars are alsoextremely effective at agitating magnetic beads in a liquid enclosedwithin for example, a 1.5 ml polypropylene microcentrifuge tube. Themicrocentrifuge tube is simply stood, or laid flat on the top surface ofthe magnetic stirrer and the speed setting adjusted to between 100-900rpm. The beads are vigorously agitated within the microcentrifuge tube,which itself is immobile. A simple microcentrifuge tube holder made offoam or plastic and set on top of the magnetic stirrer provides aconvenient means to hold the tubes at the same distance from therotating permanent magnet underneath. The beads can be collected bymoving the tube holder containing the tubes to a fixed magnetic stand.Suitable magnetic stirrers include IKA® magnetic stirrers (Aldrich,catalogue number Z40,482-9, Z40,372-5). Alternatively, magnetic stirrerswith no electric motors but rather, a series of oscillatingelectromagnets can be used effectively to thoroughly mix the magneticbeads (Aldrich, catalogue number Z31,233-5).

EXAMPLE 1 Purification of RNA from Human Blood Plasma

Various types of RNA can be purified from plasma such as the clinicallyimportant viruses HCV and HIV. The virus capsids are disrupted in thepresence of 1-methylimidazole, tetrahydrofuran (THF) and an acylatingreagent such as acetic anhydride. This mixture also leads to thedisruption of the nucleoprotein complex and consequent release of theRNA which can then be chemically modified and stabilised.

Stabilisation of the RNA analyte: To 200 μl of human plasma (EDTAcoagulation inhibitor) or serum in a 15 ml screw top polypropylenecentrifuge tube (Falcon, USA) was added 50 μl of 1-methylimidazole, themixture was mixed briefly and then 600 μl of a mixture oftetrahydrofuran and acetic anhydride (2:1 vol/vol) was added and mixedby gentle pipetting with a 1 ml pipette tip. A second addition of 600 μlof tetrahydrofuran and acetic anhydride (2:1 vol/vol) was added within 1minute of the first addition and the solution mixed again. Other typesof acylating reagents such as propionic, butanoic, pentanoic, heptanoicor benzoic anhydrides can be used as alternatives or in conjunction withacetic anhydride. Acylation reagents and methods for chemicallymodifying RNA have been described (WO/01/94626 and WO/00/75302)

Following addition of the acylation reagent, an internal control such asone composed of RNA (e.g. 8 μl of the HCV Internal Control, version 2.0,Roche Diagnostics, USA) or DNA or even a bacteriophage (Armoured RNA,Hepatitis Virus Control, Ambion RNA Diagnostics, USA) may be added.Following an incubation for 10 minutes at 37° C., the solutioncontaining the stabilised RNA can be stored for prolonged periods at upto 37° C. The RNA can also be transported and handled in its protectedform. Alternatively, the RNA can be purified immediately following the10 minute incubation at 37° C. as described below.

It has been found that with human plasma samples such as thoseoriginating from blood donations, on addition of tetrahydrofuran andacetic anhydride, the reaction turns bright fluorescent yellow, servingas a useful indicator that the reaction was successful. On standing for10 minutes the reaction turns brown.

Processing of the stabilised RNA: Following stabilisation of the RNAduring which the RNA sample can be either stored, transported orprocessed immediately (following the 10 minute incubation at 37° C.) theRNA is separated from the reaction and contaminants by differentialbinding to a solid support such as silica or hydroxylapatite in thepresence of an organic amine. Alternatively, but less preferably the RNAcan be purified by precipitation, dialysis or spin column filtration.The RNA can also be spotted and immobilised directly onto a solidsupport such as nylon (Hybond N+, Amersham Pharmacia Biotech, UK) andanalysed by hybridisation with a labelled complementary probe.

It has been found that primary amines such as ethylenediamine orethanolamine are particularly suited to reducing contamination byprotein binding to the solid phase used to bind and purify the nucleicacid, and in the case of RNA, also leads to the cleavage of theprotecting acetyl group from the 2′-OH position of the RNA. The primaryamine therefore has two useful properties (i) reducing proteincontamination and, (ii) deprotecting the RNA without leading toconsequent phosphodiester cleavage. The use of ethylenediamine andethanolamine for the deprotection of oligonucleotides has been described(Miller, P. S. et al. (1986) Biochem. 25:5092; Hogrefe et al., NucleicAcids Res (1994) 22:5492; Polushin, N. (1994) Nucleic Acids Res. 22:639;Polushin, N. (1991) Nucleic Acids Symp Ser. 24:49). However, the use ofthese deprotection reagents was limited to the removal of protectinggroups from the nucleobases of DNA and it is unexpected that our resultshave demonstrated that primary amines lead to only limited cleavage ofthe phosphodiester bond of RNA as would be expected by a strong base.

To 1.45 ml of the reaction containing the desired RNA analyte is added1.4 ml of ice cold 1-methylimidazole and the solution mixed by gentlepipetting. Then three separate aliquots of 200 μl of a prepared solutionof 1 ml of 1-methylimidazole, 1 ml of either ethanolamine or preferablyethylenediamine containing 50 μl (40 mg/ml) of phosphate treatedhydroxylapatite Type I (Chemicell, Germany) are added and the reactionincubated for 2 minutes at 25° C., before the remaining 1.45 ml of the1-methylimidazole, primary amine and beads are added and mixed. Theentire mixture is then slowly mixed using an end-over-end wheel for 10minutes at 25° C. to allow deprotection of the RNA and binding to thebeads. However, agitating the mixture is not essential. The addition ofthe amine leads to an exothermic reaction, and the amine is thereforemost easily added to the RNA containing reaction diluted into the buffer1-methylimidazole. Alternatively, silica may be used in the place ofhydroxylapatite to bind the nucleic acid.

Phosphate treatment of the beads is as follows; to 1 ml ofhydroxylapatite Type I (50 mg/ml) was added 2 ml of 0.2M sodiumphosphate (pH 7) and the beads are briefly mixed by inverting the tube.The beads are then collected with a magnet and the liquid discarded, thebead pellet is then resuspended in 2 ml of water, briefly mixed, thebeads collected and the water wash repeated once more, the beads arethen resuspnded in 1 ml of water. It has been found that hydroxylapatitebeads treated in this way bind less protein and the RNA is more easilyeluted than the non-phosphate treated beads.

The beads are then collected from the reaction using a magnetic stand(Dynal, Norway) and the liquid discarded by pouring it away from thebead pellet. The beads are then resuspended in 1 ml of 70%methanol/ethanol (1:1 vol/vol) and transferred to a fresh 2 mlpolypropylene centrifuge tube and collected using a magnetic stand(Promega, USA). The beads are washed two more times in 1 ml of 70%methanol/ethanol and then washed three times in 100 μl of water. It isimportant not to touch the beads with the pipette tip when they are inaqueous suspension because they tend to stick to plastic resulting insample loss. The RNA is now ready for storage, transport or can beimmediately eluted from the hydroxylapatite beads.

The nucleic acid can be released from the hydroxylapatite bead by avariety of means including the use of phosphate containing solutions ora divalent metal ion chelator such as EGTA. The mechanism of nucleicacid elution most probably involves a competition between the nucleicacid phosphate groups and the hydroxylapatite calcium atoms. It has beenfound that nucleotide triphosphate groups and metal ion chelators areparticularly effective at displacing nucleic acids from thehydroxylapatite.

To the nucleic acid hydroxylapatite bead mixture (approximate volume50-100 μl) is added 50 μl of a nucleotide solution, the beads agitatedby means of a magnetic stirrer set on the program 0.5 second step,(MCB1200, Dexter Magnetics, UK) for 2 minutes at 25° C., the beadscollected using the magnet, the liquid collected and then the processrepeated 3 more times and the liquid containing the nucleic acid pooled.The nucleotide solution can be a 5 mM dNTP solution such as dATP, dCTP,dGTP, TTP, dUTP or a 5 mM solution of ribose NTP such as rATP, rCTP,rGTP, rUTP or a 50 mM solution of dNDP or rNDP, dNMP or rNMP or eveninorganic pyrophosphate (sodium phosphate) as described (Sambrook etal., (1989) Molecular Cloning: A Laboratory Manual, CSH). It has beenfound that as the nucleic acid is displaced from the hydroxylapatite,the total nucleotide concentration is reduced indicating it is bindingto the hydroxylapatite in place of the eluted nucleic acid. This isimportant when calculating the final nucleotide concentration fordownstream analytical procedures such as RT-PCR.

Alternatively, and preferably, the nucleic acid can be eluted from thehydroxylapatite using calcium ion chelators such as CDTA(trans-1,2-Diaminocyclohexane-N,N,N′,N′-tetraacetic acid), EDTA(Etylenediamine tetraacetic acid), EGTA(Etylenenglycol-O,O′-bis(2-aminoethyl)-N,N,N′,N′-tetraacetic acid), DTPA(Diethylenetriamine pentaacetic acid), HEDTA(N-(2-Hydorxyethyl)ethylenediamine-N,N,N′-triacetic acid), NTA(Nitrilotriacetic acid), TTHA(Triethylenetetramine-N,N,N′,N″,N′″,N″″-hexaacetic acid), Dimethyl-BAPTA(Molecular Probes, USA) or BAPTA(Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid). To the nucleicacid hydroxylapatite bead mixture (approximate volume 50-100 μl) isadded 50 μl of a 10 mM chelator solution, the beads agitated by means ofa magnetic stirrer set on the program 0.5 second step, (MCB1200, DexterMagnetics, UK) for 2 minutes at 25° C., the beads collected using themagnet, the liquid collected and then the process repeated 7 more timesand the liquid containing the nucleic acid pooled. It has been foundthat 75% of the nucleic acid is eluted between the 5th to the 8thelution (200-400 μl) of a 10 mM solution of EGTA. By comparison, it hasalso been found that DNA is eluted almost equally into the 1st to the7th 50 μl elution volume.e as determined by both 32P labelled DNA tracerstudies and PCR amplification of the eluted DNA.

The eluted RNA and the material used to elute it from thehydroxylapatite can either be used directly in an analytical proceduresuch as hybridisation, or in an enzymatic assay such as reversetranscription-PCR. Whilst phosphate containing elution solutions workefficiently to elute nucleic acids from hydroxylapatite, they are notideal because excess phosphate tends to interact and therefore reducethe final concentration of metal ions in enzymatic reactions leading toreduced yields. Therefore it is preferred to use metal ion chelatorssuch as EGTA which have higher specificity for certain types of divalentmetal ions. It has been found that when 10 mM EGTA is used to elute thenucleic from the hydroxylapatite, the final EGTA concentration isapproximately 4-5 mM. EGTA has a lower stability constant (logK 5.21)for magnesium ions than EDTA (logK 8.69), therefore it has been foundthat the presence of at least 8 mM (final EGTA concentration) in aRT-PCR reaction does not detectably alter the amplicon yield. Thereforefor assays employing magnesium as a divalent metal cation at, forexample 2 mM or more, such as MULV, AMV and Taq DNA polymerases, EGTA isa good choice for eluting the nucleic acid. It is also possible to use aminimum amount of EGTA necessary to elute the RNA so that all the EGTAin solution is effectively captured by the magnetic-hydroxylapatiteleaving the RNA essentially EGTA free as set out in example 29 However,it has also been found that EGTA/nucleic acid solutions tend to chelatemanganese ions such as those solutions employed in commercial diagnosticRT-PCR assays for example the Amplicor HCV Test, version 2.0 (RocheDiagnostics, USA) leading to reduced amplification yield when too muchof the EGTA/nucleic acid solution is added. It has been found forexample that the addition of 6 or more of the EGTA/nucleic acid solutionto a standard 100 μl Amplicor HCV Test, version 2.0 leads to reducedOD660 nm readings indicating that the amplification efficiency isreduced.

Where the assay employs manganese ions, there are several practicalsolutions to the problem of chelation and therefore inhibition of theamplification. Firstly the chelator can be removed using a differentialbinding process whereby the chelator molecule such as EGTA can diffusethrough a semi-permeable material/barrier such as a gel, membrane,polymer or pore and bind to a chelator binding material such ashydroxylapatite. It has been found that suitable methods for removingsmall chelators from a nucleic acid solution are (i) mixinghydroxylapatite beads with 0.2-0.8% molten agarose or preferablypolymerising acrylamide with hydroxylapatite and allowing it to solidifythereby entombing the hydroxylapatite. The EGTA/nucleic acid solution isthen placed in contact with the gel and the EGTA (or other chelator)allowed to diffuse into the gel, whilst the gel prevents the larger RNAmolecules from contacting the beads and therefore from being lost. Ithas been found that 20 minutes is sufficient to remove 90% of the EGTAfrom a 50 μl volume of 5 mM EGTA solution at ambient temperature whenplaced in contact with a 1 cm2 area of 100 μl of the gel containing 0.5mg hydroxylapatite (type I, Chemicell, Germany). The remaining liquidcontaining the nucleic acid is then added to the assay. Alternatively,the hydroxylapatite beads can be coated in a polymer such as a heparin,cellulose, starch or dextran to provide a semi-permeable barrier to thenucleic acid whilst still allowing the chelator molecules to bind thehydroxylapatite. A 200 μl volume of the polymer solution (10%) isconveniently added to 0.5 mg hydroxylapatite (type I, Chemicell,Germany) beads and allowing the solution to dry completely at 90° C. for1 hour.

Another method (ii) for removing the chelator is to filter the nucleicacid/chelator solution. Although many filtration methods exist forselectively removing contaminants from nucleic acids, such as sizeexclusion chromatography and silica membranes, a preferred method is touse centrifugal or pressure filter devices, also known asultrafiltration such as those known as Centricon, Centriprep, Centriplusand Centricon Plus® manufactured by Millipore (USA). In particular ithas been found that Microcon centrifugal devices are well suited forthis purpose. These devices use regenerated cellulose with various poresizes with molecular weight cut offs from 3 000-100 000 daltons. Thepreferred size is 50 000 daltons molecular weight cut off (Part No.42416, Millipore, USA) so that nucleic acids more than 200 nucleotidesin length are retained by the membrane, whilst the chelator such as EGTApasses through the membrane and are discarded. The nucleic acid can beremoved from the hydroxylapatite using a chelator, a phosphatecontaining solution, or a calcium binding salt. An advantage of thefiltration method is that not only are contaminants removed but thenucleic acid is concentrated. This is particularly advantageous when theelution solution containing the nucleic acid is more than 100 μl becausethe final filtered and concentrated volume can be 10 μl so that theentire sample can be added to the analytical test. For example with theHCV Amplicor test v2.0 (Roche Diagnostics, USA) the maximum volume thatmay be added to each test is 50 μl, whilst the eluted nucleic acidvolume may be as much as 0.5 ml. The filtration therefore serves twopurposes; to remove the contaminants and to concentrate the analyte.

The preferred method of filtration is as follows. Using 10 mM EGTA asthe elution solution, the first 400 μl of the elution from thehydroxylapatite beads is collected and pooled. The elution can either be8 separate elutions of 50 μl with 2 minute elution steps using mixing asdescribed above (MCB1200, Dexter Magnetics, USA) or 1 elution with 400μl of 10 mM EGTA with a ten minute elution with mixing. It is alsopossible to add a smaller volume of more concentrated elution solutionsuch as μl of 20 mM EGTA and mixing for 10 minutes at room-temperature.In any case, the final elution volume is conveniently no more than 400μl which is the maximum volume accomodated by the Microcon filtrationdevice (Part No. 42416, Millipore, USA). The device is then centrifugedat 12 000 g for 20 minutes (until dryness), 400 μl of water added andthe device spun again at 12 000 g for 20 minutes (until dryness). Then25-50 μl of water is added to the device to recuperate the nucleic acidthe cup inverted in a 2 ml fresh tube and centrifuged for 10 seconds at2500 g. The nucleic acid can then be used in the assay. Proteins thatare larger than 50 000 daltons are also retained with the nucleic acid,however, it has been found that no detectable inhibition occured duringRT-PCR(HCV Amplicor v2.0, Roche Diagnostics, USA) with RNA prepared inthis manner from blood plasma. Alternatively the nucleic acid can beeluted from the hydroxylapatite using 400 μl of 100-500 mM SodiumPhosphate (pH 7) and mixing for 10 minutes at room temperature. Thephosphate/nucleic acid solution can then be filtered using a Microconfiltration device as described above.

Yet another method (iii) for relieving the inhibitory effects of thechelator and in particular the EGTA solution is to ‘neutralise’ thecapacity of the chelator to bind the manganese or magnesium. This can beaccomplished in a number of ways. The first method is to add a metal ionthat has a higher stability constant for the chelator than the metal ionnecessary for the assay. Unfortunately Mn ions have a particularly highstability constant (12.3) with EGTA compared with Mg ions (5.21).Therefore it is preferable to choose metal ions with stability constantshigher than Mn (12.3) so that the added metal ion competes effectivelywith the Mn for the chelator. Suitable metal ions are Fe(III) (20.3), Cu(II) (17.8), Co (II) (12.30) and Zn (II) (14.5). These metal ions can beadded to the chelator containing solution as a salt such as the chlorideor acetate. Although other metal ions also have high stability constantsfor EGTA, such as Ni, Hg and Cd, their toxicity would preclude theirroutine use. It has been found that adding 3.5-7 mM final concentrationof CoCl2 to a 100 μl standard HCV Amplicor (Roche Diagnostics, USA)reaction containing 25 μl of RNA eluted from the hydroxylapatite beadsusing 10 mM EGTA effectively removes the inhibitory effects of the EGTAallowing amplification of the HCV analyte RNA. The most preferable CoCl2concentrations are 4.5 mM and 6 mM. Alternatively, CuCl2 or FeCl3 canalso be used in the range 3.5-7 mM but are less effective.

It is also possible to use an additional amount of Mn in the analyticalprocedure to replace the Mn bound to the chelator. It has been foundthat adding 1-3 mM, or preferably 1.5 mM Manganese acetate to a 100 μlstandard HCV Amplicor reaction containing 25 μl of RNA eluted from thehydroxylapatite beads using 10 mM EGTA effectively removes theinhibitory effects of the EGTA allowing amplification of the HCV analyteRNA. Therefore it is not strictly necessary that the metal ion has astability constant higher than Mn in order to overcome the inhibition.However, 4.5 mM CoCl2 is preferred in the HCV Amplicor test as describedabove.

RNA can also be eluted directly from the hydroxylapatite beads into a 5×solution of the RT-PCR buffer (250 mM bicine/KOH buffer pH8.2, 575 mMpotassium acetate, 40% glycerol (w/v) used for Tth DNA polymerase andthen added to the other reaction components (Roche Amplicor HCV v2.0)prior to amplification.

If silica particles or membranes are used for capturing the nucleicacid, then an equal weight of silica such as Qiaex II (Qiagen, Germany)or Magnisil (Promega, USA) is added in the place of hydroxylapatitebeads. In this case the silica beads, following the deprotectionreaction with the primary amine are washed in four rinses of 1 ml of 70%ethanol and then the nucleic acid is eluted in 200 μl of water followingincubation for 10 minutes at 37° C.

EXAMPLE 2 Purification of DNA from Human Blood Plasma

Various types of DNA can be purified from plasma such as the clinicallyimportant virus HBV or bacteria. The viral or bacterial particles aredisrupted in the presence of 1-methylimidazole, tetrahydrofuran (THF)and an acylating reagent such as acetic anhydride. This mixture alsoleads to the disruption of the nucleoprotein complex and consequentrelease of the DNA which can then be chemically modified and stabilised.

Methods for purifying DNA are substantially similar to methods forpurifying RNA as set out in Example 1 above. However, unlike RNA, ds DNA(300 bp) tends to elute from the first to the 7th addition of 50 μl of10 mM EGTA, therefore all fractions of the elution should be kept.

EXAMPLE 3 Purification of DNA and RNA Simultaneously from Human Plasma

Both DNA and RNA can be purified from plasma at the same time, allowingsimultaneous testing of both clinically important RNA sources such asHCV and HIV as well as DNA sources such as HBV from the same elutednucleic acid sample. The viral or bacterial particles are disrupted inthe presence of 1-methylimidazole, tetrahydrofuran (THF) and anacylating reagent such as acetic anhydride. This mixture also leads tothe disruption of the nucleoprotein complex and consequent release ofthe DNA which can then be chemically modified and stabilised.

Methods for purifying DNA and RNA are substantially similar to methodsset out in Example 1 and 2 above.

EXAMPLE 4 Purification of DNA and/or RNA from Whole Human Blood

If present, both DNA and RNA can be purified from whole blood at thesame time, allowing testing of both clinically important RNA sourcessuch as HCV and HIV as well as DNA sources such as HBV from the sameeluted nucleic acid sample.

Methods for purifying DNA and/or RNA are substantially similar toExample 1 above, except that 200 μl, instead of 50 μl of1-methylimidazole is added to 200 μl of blood. It has been found thatthe addition of 200 μl, instead of 50 μl of 1-methylimidazole helps tosolubilise the cellular components present in the blood as theerythrocytes and white blood cells. Although it has been found that 50μl of 1-methylimidazole also works well, the mixture of1-methylimidazole, tetrahydrofuran and acetic anhydride tends to formlarge clumps which only dissolve once the deprotection reagent (e.g.ethylenediamine) is added.

Alternatively, 200 μl of blood can be dissolved into 1 ml of NMP:aceticanhydride (2:1 vol:vol) and then 3 ml of 1-methylimidazole added,followed by an incuabation of 10 min. at 37° C.

EXAMPLE 5 Purification of DNA and/or RNA from Cells

Both DNA and RNA can be purified from cells at the same time, allowingpurification of both DNA and cellular RNA such as rRNA, tRNA and mRNA.

Methods for purifying DNA and/or RNA are substantially similar toExample 1 above, except that 200 μl, instead of 50 μl of1-methylimidazole is added to 200 μl of a centrifuge collected pellet (3000 g×10 minutes) of 1 million tissue culture cells. It has been foundthat the addition of 200 μl, instead of 50 μl of 1-methylimidazole helpsto solubilise the cellular components present. Although it has beenfound that 50 μl of 1-methylimidazole also works well, the mixture of1-methylimidazole, tetrahydrofuran and acetic anhydride tends to formlarge clumps which only dissolve once the deprotection reagent (e.g.ethylenediamine) is added.

Alternatively, 50 mg of tissue or organ sample can be disrupted using adounce homegeniser or sonicator in the presence of 200 μl of1-methylimidazole, then the mixture immediately added to 600 μl ofTHF/acetic anhydride (2:1 vol:vol), mixed before a second addition of600 μl of THF/acetic anhydride (2:1 vol:vol). Following a 10 minuteincubation at 37° C. the RNA is purified identically as for Example 1starting at the addition of 1.4 ml of 1-methylimidazole.

It has been found that a mixture of any one of the solventsN-methylpyrrolidinone, Formyl morpholine or Di-methyl imidazolidone withacetic anhydride (2:1 vol:vol) efficiently lyses only the cell membraneof tissue culture cells but not the nuclear membrane. Thereforecytoplasmic nucleic acids are preferrentially released and purifiedusing these solvents. This is a useful method for reducing contaminationwith nuclear located genomic DNA.

EXAMPLE 6 Purification of DNA from Human Faeces or Urine

The method is as described as for example 1 except that the 200 μl ofplasma is replaced by 200 μl of urine or 200 μl of faeces diluted inwater to 10%.

EXAMPLE 7 Deprotection of Acetyl Modified RNA Using Gaseous Ammonia

Preparation of modified RNA. To 40 μl of tetrahydrofuran containing 16%1-methylimidazole was added 100 ng-1 μg of RNA, then 2-41 μl of aceticanhydride was mixed into the reaction and incubated for 10 minutes at37° C. To the reaction was added 10 μl of magnetic hydroxylapatite Type1 (40 mg/ml) (Chemicell GmbH, Berlin, Germany) or magnetic silicaparticles (<<Magnisil>>, Promega, USA) and mixed for 10 minutes at 25°C. The beads were then collected using a magnetic stand (Promega, USA)and the liquid discarded. The beads were washed once with 95% ethanoland allowed to air dry for 5 minutes at 25° C.

Deprotection of the modified RNA. The open tube containing thebead-modified RNA mixture was placed in a 50 ml screw top polypropylenetube and a pipe inserted into a hole through the cap of the 50 ml tube.100 ml of 38% ammonium hydroxide was heated to 50° C. in a side armflask, the ammonia vapour was allowed to exit the flask and enter aseparate side arm glass flask to allow condensation of any water vapour.The dried ammoni gas was then allowed to enter the 50 ml tube containingthe beads by means of a flexible plastic pipe. The screw cap of the 50ml tube was loosely closed allowing ammonia gas to enter the tube andthen exit to be replaced by fresh ammonia.

The acetylated RNA on the beads was subjected to the ammonia for 5-60minutes, after which the beads were washed once in water and then elutedwith 10 mM EDTA or EGTA (hydroxylapatite) or the deprotected RNA waseluted directly into 50 μl water (silica beads). It was found tht thetime for deprotection varied according to the amount of ammonia producedduring heating, 60 minutes usually being sufficient to removesubstantially all acetyl groups from the RNA. The deprtotected RNA canthen be used for various downstream applications such as RT-PCR andhybridisation.

EXAMPLE 8 Deprotection of Acetyl Modified RNA Using Ethylenediamine

Preparation of modified RNA. To 40 μl of tetrahydrofuran containing 16%1-methylimidazole was added 100 ng-1 μg of RNA, then 2-4 μl of aceticanhydride was mixed into the reaction and incubated for 10 minutes at37° C. To the reaction was added 10 μl of magnetic hydroxylapatite Type1 (40 mg/ml) (Chemicell GmbH, Berlin, Germany) or magnetic silicaparticles (<<Magnisil>>, Promega, USA) and mixed for 10 minutes at 25°C. The beads were then collected using a magnetic stand (Promega, USA)and the liquid discarded.

To the wet beads was added 50-500 μl of ethylenediamine (Fluka cat. No.03550, France) and the beads stirred briefly and incubated for 1-60minutes at 25° C. The beads were then collected with a magnet and theliquid discarded. The beads were washed twice with 200 μl of 70%ethanol/methanol (1:1) once with water and then the deprotected RNA waseluted into 100 μl of 10 mM EDTA or EGTA by incubating for 10 minutes at25° C. It was found that 1 minute with 300 μl of ethylenediamine was notsufficient to fully deprotect the RNA as assayed using an in vitrotranscript labelled with 32P and a 5% acrylamide sequencing gel. Therate of migration is proportional to the amount of deprotection andsequencing gels serve as a useful measure of the amount of deprotectionthat has occured. After 5 minutes the RNA was over 90% deprotected andafter 15 minutes the RNA was completely deprotected. Increasing thedeprotection time to 60 minutes at 25° C. did not lead to any detectableRNA degradation.

EXAMPLE 9 Deprotection of Acetyl Modified RNA Using Ethanolamine

Preparation of modified RNA. To 40 μl of tetrahydrofuran containing 16%1-methylimidazole was added 100 ng-1 μg of RNA, then 2-4 μl of aceticanhydride was mixed into the reaction and incubated for 10 minutes at37° C. To the reaction was added 10 μl of magnetic hydroxylapatite Type1 (40 mg/ml) (Chemicell GmbH, Berlin, Germany) or magnetic silicaparticles ((<<Magnisil>>), Promega, USA) and mixed for 10 minutes at 25°C. The beads were then collected using a magnetic stand (Promega, USA)and the liquid discarded.

To the beads that were not dried from the previous protection reaction,was added 50-500 μl of ethanolamine (Fluka cat. No. 02400, France) andthe beads stirred briefly and incubated for 1-60 minutes at 25° C. Thebeads were then collected with a magnet and the liquid discarded. Thehydroxylapatite beads were washed twice with 200 μl of 70%ethanol/methanol (1:1), once with water and then the deprotected RNA waseluted into 100 μl of 10 mM EDTA or EGTA by incubating for 10 minutes at25° C. The silica beads were washed twice in 200 μl of Wash solution PE(Qiagen, Germany) and the deprotected RNA eluted in 100 μl of water.

It was found that 1 minute with 300 μl of ethylenediamine was notsufficient to fully deprotect the RNA as assayed using an in vitrotranscript labelled with 32P and a 5% acrylamide sequencing gel. Therate of migration is proportional to the amount of deprotection andsequencing gels serve as a useful measure of the amount of deprotectionthat has occured. After 5 minutes the RNA was over 70% deprotected andafter 15 minutes the RNA was was over 90% deprotected. Completedeprotection occured after 30 minutes at 25° C. Increasing thedeprotection time to 60 minutes at 25° C. led to detectable degradationof a 1700 nucleotide RNA molecule but relatively little degradation of a250 nucleotide molecule as determined by sequencing gel analysis.Therefore deprotection with ethanolamine was slightly slower than withethylelenediamine and also led to more RNA degradation. However, thismay have been at least in part due to the purity of the ethanolamine.

EXAMPLE 10 Deprotection of Acetyl Modified RNA Using Mixtures ofEthanolamine and Ethylenediamine

Preparation of modified RNA. To 40 μl of tetrahydrofuran containing 16%1-methylimidazole was added 100 ng-1 μg of RNA, then 2-4 μl of aceticanhydride was mixed into the reaction and incubated for 10 minutes at37° C. To the reaction was added 10 μl of magnetic hydroxylapatite Type1 (40 mg/ml) (Chemicell GmbH, Berlin, Germany) or magnetic silicaparticles (<<Magnisil >>, Promega, USA) and mixed for 10 minutes at 25°C. The beads were then collected using a magnetic stand (Promega, USA)and the liquid discarded.

To the wet beads was added 50-500 μl of a mixture of ethanolamine andethylenediamine (1:1), (Fluka, France) and the beads stirred briefly andincubated for 1-60 minutes at 25° C. The beads were then collected witha magnet and the liquid discarded. The hydroxylapatite beads were washedtwice with 200 μl of 70% ethanol/methanol (1:1) once with water and thenthe deprotected RNA was eluted into 100 μl of 10 mM EDTA or EGTA byincubating for 10 minutes at 25° C. The silica beads were washed twicein 200 μl of Wash solution PE (Qiagen, Germany) and the deprotected RNAeluted in 100 μl of water.

EXAMPLE 11 Deprotection of Acetyl Modified RNA Using Ethanolamine orEthylenediamine at Increased Temperatures

Preparation of modified RNA. To 40 μl of tetrahydrofuran containing 16%1-methylimidazole was added 100 ng-1 μg of RNA, then 2-4 μl of aceticanhydride was mixed into the reaction and incubated for 10 minutes at37° C. To the reaction was added 10 μl of magnetic hydroxylapatite Type1 (40 mg/ml) (Chemicell GmbH, Berlin, Germany) or magnetic silicaparticles (<<Magnisil>>, Promega, USA) and mixed for 10 minutes at 25°C. The beads were then collected using a magnetic stand (Promega, USA)and the liquid discarded.

To the wet beads was added 100 μl of ethylenediamine or ethanolamine andthe beads stirred briefly and incubated for 20 minutes at 37, 45 and 55°C. The beads were then collected with a magnet and the liquid discarded.The beads were washed twice with 200 μl of 70% ethanol/methanol (1:1)once with water and then the deprotected RNA was eluted into 100 μl of10 mM EDTA or EGTA by incubating for 10 minutes at 25° C.

The amount of deprotection of the RNA as assayed using an in vitrotranscript labelled with 32P and a 5% acrylamide sequencing gel. Therate of migration is proportional to the amount of deprotection andsequencing gels serve as a useful measure of the amount of deprotectionthat has occured. It was found that with ethanolamine at 55, 45 or 37°C. there was significant degradation of the RNA, whilst withethylenediamine at 55° C. there was limited degradation, bothdeprotection at 45 or 37° C. did not lead to detectable RNA degradation.Therefore deprotection with ethanolamine is best achived at 25° C.whilst with ethylenediamine, deprotection can be carried out up to 45°C.

EXAMPLE 12 Deprotection of Acetyl Modified RNA Using Ethanolamine orEthylenediamine in Alcohol

Preparation of modified RNA. To 40 μl of tetrahydrofuran containing 16%1-methylimidazole was added 100 ng-1 μg of RNA, then 2-4 μl of aceticanhydride was mixed into the reaction and incubated for 10 minutes at37° C. To the reaction was added 10 μl of magnetic hydroxylapatite Type1 (40 mg/ml) (Chemicell GmbH, Berlin, Germany) or magnetic silicaparticles (<<Magnisil>>, Promega, USA) and mixed for 10 minutes at 25°C. The beads were then collected using a magnetic stand (Promega, USA)and the liquid discarded.

To the wet beads was added 100 μl of ethylenediamine or ethanolamine and100 μl of methanol/ethanol (1:1) and the beads stirred briefly andincubated for 20 minutes at 37, 45 and 55° C. The beads were thencollected with a magnet and the liquid discarded. The beads were washedtwice with 200 μl of 70% ethanol/methanol (1:1) once with water and thenthe deprotected RNA was eluted into 100 μl of 10 mM EDTA or EGTA byincubating for 10 minutes at 25° C.

It was found that the addition of alcohol was not beneficial to thedeprotection reaction, indeed the rate of deprotection was reducedwhilst the amount of RNA degradation was increased.

EXAMPLE 13 Deprotection of Acetyl Modified RNA Using Ethanolamine orEthylenediamine with a Strong Alkali

Preparation of modified RNA. To 40 μl of tetrahydrofuran containing 16%1-methylimidazole was added 100 ng-1 μg of RNA, then 2-4 μl of aceticanhydride was mixed into the reaction and incubated for 10 minutes at37° C. To the reaction was added 10 μl of magnetic hydroxylapatite Type1 (40 mg/ml) (Chemicell GmbH, Berlin, Germany) or magnetic silicaparticles (<<Magnisil>>, Promega, USA) and mixed for 10 minutes at 25°C. The beads were then collected using a magnetic stand (Promega, USA)and the liquid discarded.

To the wet beads was added 100 μl of ethanolamine plus either 101 μl of10% ammonium hydroxide, 10 μl of 10 mM NaOH or 10 μl of 50 mM NaOH andthe beads stirred briefly and incubated for 20 minutes at 37° C. Thebeads were then collected with a magnet and the liquid discarded. Thebeads were washed twice with 200 μl of 70% ethanol/methanol (1:1) oncewith water and then the deprotected RNA was eluted into 100 μl of 10 mMEDTA or EGTA by incubating for 10 minutes at 25° C.

It was found that the addition of either ammonium hydroxide or NaOH didnot increase the amount of deprotection compared with ethanolamine alonebu did increase the amount of RNA degradation.

EXAMPLE 14 Deprotection of Acetyl Modified RNA Using Ethanolamine andEthylenediamine in the Presence of Water

Preparation of modified RNA. To 40 μl of tetrahydrofuran containing 16%1-methylimidazole was added 100 ng-1 μg of RNA, then 2-4 μl of aceticanhydride was mixed into the reaction and incubated for 10 minutes at37° C. To the reaction was added 10 μl of magnetic hydroxylapatite Type1 (40 mg/ml) (Chemicell GmbH, Berlin, Germany) or magnetic silicaparticles (<<Magnisil>>, Promega, USA) and mixed for 10 minutes at 25°C. The beads were then collected using a magnetic stand (Promega, USA)and the liquid discarded.

To the wet beads was added 50 μl of ethanolamine, 10 μl of water and 50μl of methanol and the beads stirred briefly and incubated for 5 minutesat 25° C. The beads were then collected with a magnet and the liquiddiscarded. The beads were washed twice with 200 μl of 70%ethanol/methanol (1:1) once with water and then the deprotected RNA waseluted into 100 μl of 10 mM EDTA or EGTA by incubating for 10 minutes at25° C.

It was found that the addition of water did not alter the amount ofdeprotection compared with ethanolamine and alcohol alone and did notincrease the amount of RNA degradation. It is therefore not essentialthat water be removed from solutions and the beads prior todeprotection.

EXAMPLE 15 Alternative Means to Remove RNA from Hydroxylapatite Beads

It has been found that either protected or deprotected RNA can beremoved from hydroxylapatite beads by simply loading the bead-RNAcomplex into either a well of a 0.5×TAE agarose gel or a well of a 1×TBEsequencing gel and applying an electric field through the wellcontaining the beads. The protected or deprotected RNA readilydissociates from the hydroxylapatite beads and electrophoresis into thegel where it can be either collected or analysed by means for example ofEtBr or a radioactive label. This is a very convenient means to detachRNA from hydroxylapatite.

Protected or deprotected RNA may also be separated from hydroxylapatiteby inserting 2 wires (anode and cathode) without them touching into atube containing beads in 100 l of water or buffer and applying a lowvoltage such as 5-50V for 5 minutes. The RNA can be recuperated from theliquid phase.

EXAMPLE 16 RT-PCR Amplification of Deprotected RNA

Preparation of modified template RNA. To 100 μl of a mixture oftetrahydrofuran/acetic anhydride (2:1 vol/vol) was added 14 μl of1-methylimidazole and 2 μg of BMV RNA Promega, USA), the mixture stirredand incubated for 2 minutes at 25° C. To the reaction was added 60 μl of1-butanol and then 20 μl of magnetic hydroxylapatite Type 1 (40 mg/ml)(Chemicell GmbH, Berlin, Germany) and mixed for 3 minutes at 25° C. Thebeads were then collected using a magnetic stand (Promega, USA), washedonce in 200 μl of 70% methanol/ethanol (1:1) and the liquid discarded.

To the wet beads was added either 100 μl of ethanolamine or 100 μl ofethylenediamine and the beads stirred briefly and incubated for 20minutes at 37° C. The beads were then collected with a magnet and theliquid discarded. The beads were washed twice with 200 μl of 70%ethanol/methanol (1:1) once with water and then the deprotected RNA waseluted into 100 μl of 10 mM EGTA by incubating for 10 minutes at 25° C.

Reverse Transcription. 25 ng of the deprotected BMV RNA was added to a20 μl reaction mixture containing the following final componentconcentrations: 200 mM Tris-HCl (pH 8.4 at 24° C.), 75 mM KCl, 2.5 mMMgCl₂, 10 mM DTT, 1 mM dNTP's, 60 ng of oligonucleotide primer BMV R(GAGCCCCAGCGCACTCGGTC) and MULV RNase H⁻ (Promega, cat no. M3682, USA).Water was used to bring the final volume to 20 μl. The reaction wasallowed to proceed for 20 minutes at 37° C., 20 minutes at 42° C. and 20minutes at 50° C. PCR Amplification. The PCR was carried out in a finalvolume of 25 μl with final concentration of 15 mM Tris-HCl pH 8.8, 60 mMKCl, 2.5 mM MgCl₂, 400 μM each dNTP, 10 pmol of each primer BMV F(CTATCACCAAGATGTCTTCG) and BMV R and 1 unit Taq DNA polymerase (RocheMolecular, France). To the PCR mix was added 2 μl of cDNA generated fromthe deprotected BMV RNA. Cycle parameters were 94° C.×8 sec, 58° C.×8sec and 72° C.×15 sec for 30 cycles. The 250 bp PCR products werevisualised following gel electrophoresis and staining with EtBr.

Excellent amplification was observed with both ethylenediamine andethanolamine deprotected RNA, indeed no significant differences could beseen in the yield of PCR product between protected-deprotected RNAcompared with an untreated RNA control indicating that no substantialdegradation of the RNA occured during deprotection.

EXAMPLE 17 Hybridisation of Deprotected RNA

Preparation of modified template RNA. To 100 μl of a mixture oftetrahydrofuran/acetic anhydride (2:1 vol/vol) was added 14 μl of1-methylimidazole and 2 μg of BMV RNA (Promega, USA), the mixturestirred and incubated for 2 minutes at 25° C. To the reaction was added60 μl of 1-butanol and then 20 μl of magnetic hydroxylapatite Type 1 (40mg/ml) (Chemicell GmbH, Berlin, Germany) and mixed for 3 minutes at 25°C. The beads were then collected using a magnetic stand (Promega, USA),washed once in 200 μl of 70% methanol/ethanol (1:1) and the liquiddiscarded.

To the wet beads was added either 100 μl of ethanolamine or 100 μl ofethylenediamine and the beads stirred briefly and incubated for 20minutes at 37° C. The beads were then collected with a magnet and theliquid discarded. The beads were washed twice with 200 μl of 70%ethanol/methanol (1:1) once with water and then the deprotected RNA waseluted into 100 μl of 10 mM EGTA by incubating for 10 minutes at 25° C.

Immobilisation of deprotected RNA. 100, 50 or 25 ng of the deprotectedBMV RNA was added to a 20 μl reaction mixture containing the followingfinal component concentrations: 200 mM Tris-HCl (pH 8.4 at 24° C.), 75mM KCl, 2.5 mM MgCl₂, 10 mM DTT, 1 mM dNTP's, 60 ng of oligonucleotideprimer BMV R (GAGCCCCAGCGCACTCGGTC) and MULV RNase H⁻ (Promega, cat no.M3682, USA). Water was used to bring the final volume to 20 μl. Thereaction was allowed to proceed for 20 minutes at 37° C., 20 minutes at42° C. and 20 minutes at 50° C. PCR Amplification. The PCR was carriedout in a final volume of 25 μl with final concentration of 15 mMTris-HCl pH 8.8, 60 mM KCl, 2.5 mM MgCl₂, 400 M each dNTP, 10 pmol ofeach primer BMV F (CTATCACCAAGATGTCTTCG) and BMV R and 1 unit Taq DNApolymerase (Roche Molecular, France). To the PCR mix was added 2 μl ofcDNA generated from the deprotected BMV RNA. Cycle parameters were 94°C.×8 sec, 58° C.×8 sec and 72° C.×15 sec for 30 cycles. The 250 bp PCRproducts were visualised following gel electrophoresis and staining withEtBr.

Excellent amplification was observed with both ethylenediamine andethanolamine deprotected RNA, indeed no significant differences could beseen in the yield of PCR product between protected-deprotected RNAcompared with an untreated RNA control indicating that no substantialdegradation of the RNA occured during deprotection.

EXAMPLE 18 Deprotection of Propanoyl Modified RNA Using Ethylenediamine

Preparation of modified RNA. To 40 μl of tetrahydrofuran containing 16%1-methylimidazole was added 100 ng-1 μg of RNA, then 2-4 μl of propionicanhydride (Fluka cat. No. 81942) was mixed into the reaction andincubated for 10 minutes at 37° C. To the reaction was added 10 μl ofmagnetic hydroxylapatite Type 1 (40 mg/ml) (Chemicell GmbH, Berlin,Germany) or magnetic silica particles (<<Magnisil>>, Promega, USA) andmixed for 10 minutes at 25° C. The beads were then collected using amagnetic stand (Promega, USA) and the liquid discarded.

To the wet beads was added 50-500 μl of ethylenediamine (Fluka cat. No.03550, France) and the beads stirred briefly and incubated for 1-60minutes at 25° C. The beads were then collected with a magnet and theliquid discarded. The beads were washed twice with 200 μl of 70%ethanol/methanol (1:1) once with water and then the deprotected RNA waseluted into 100 μl of 10 mM EDTA or EGTA by incubating for 10 minutes at25° C. It was found that 1 minute with 300 μl of ethylenediamine was notsufficient to fully deprotect the RNA as assayed using an in vitrotranscript labelled with 32P and a 5% acrylamide sequencing gel. Therate of migration is proportional to the amount of deprotection andsequencing gels serve as a useful measure of the amount of deprotectionthat has occured. After 5 minutes the RNA was over 90% deprotected andafter 15 minutes the RNA was completely deprotected. Increasing thedeprotection time to 60 minutes at 25° C. did not lead to any detectableRNA degradation.

EXAMPLE 19 Deprotection of Acetyl Modified RNA Using Polymer BoundEthylenediamine

Ethylenediamine and several other types of primary amines arecommercially available bound to a solid (polymer) support. Such primaryamines are suitable for deprotecting acetylated RNA. They provide aconvenient method for removing the deprotection reagent from thereaction once the deprotection is complete.

Preparation of modified RNA. To 40 μl of tetrahydrofuran containing 16%1-methylimidazole was added 100 ng-1 μg of RNA, then 2-4 μl of aceticanhydride (Fluka cat. No. 81942) was mixed into the reaction andincubated for 10 minutes at 37° C. To the reaction was added 10 μl ofmagnetic hydroxylapatite Type 1 (40 mg/ml) (Chemicell GmbH, Berlin,Germany) or magnetic silica particles (<<Magnisil>>, Promega, USA) andmixed for 10 minutes at 25° C. The beads were then collected using amagnetic stand (Promega, USA) and the liquid discarded. The acetylatedRNA on the hydroxylapatite beads were eluted into 200 μl of 10 mM EDTAor EGTA by incubating for 10 minutes at 25° C. The chelator/acetylatedRNA solution (200 μl) was then added to 100 mg of ethylenediamine beads(Sigma-Aldrich-Alrdich Part No. 54,748,4, USA) and incubated for 1 hr at37° C. The beads are conveniently removed by centrifugation (15 000 rpmfor 10 seconds) or filtration (Microcon device, Millipore, USA) leavingthe deprotected RNA in solution.

EXAMPLE 20 Deprotection of Acetyl Modified RNA Using Propylenediamine

Preparation of modified RNA. To 40 μl of tetrahydrofuran containing 16%1-methylimidazole was added 100 ng-1 μg of RNA, then 2-4 μl of aceticanhydride was mixed into the reaction and incubated for 10 minutes at37° C. To the reaction was added 10 μl of magnetic hydroxylapatite Type1 (40 mg/ml) (Chemicell GmbH, Berlin, Germany) or magnetic silicaparticles (<<Magnisil >>, Promega, USA) and mixed for 10 minutes at 25°C. The beads were then collected using a magnetic stand (Promega, USA)and the liquid discarded.

To the beads that were not dried from the previos protection reaction,was added 50-500 μl of propylenediamine (Fluka cat. No. 82250, France)and the beads stirred briefly and incubated for 1-60 minutes at 25-37°C. The beads were then collected with a magnet and the liquid discarded.The hydroxylapatite beads were washed twice with 200 μl of 70%ethanol/methanol (1:1), once with water and then the deprotected RNA waseluted into 100 μl of 10 mM EDTA or EGTA by incubating for 10 minutes at25° C. The silica beads were washed twice in 200 μl of Wash solution PE(Qiagen, Germany) and the deprotected RNA eluted in 100 μl of water.

EXAMPLE 21 Deprotection of Acetyl Modified RNA Using Diethylenetriamineor Tetraethylenetriamine

To 200 μl of human plasma or serum containing the nucleic acid to bestabilised and purified was added 50 μl of 1-methylimidazole and brieflymixed. To this mixture was added 1 ml of N-methylpyrrolidinone/aceticanhydride (2:1 vol:vol), mixed briefly and incubated for 10 minutes at26-37° C. To the mixture was then added 1 ml of 1-methylimidazole, mixedand incubated for 2 minutes at 26° C. and then 1.6 ml of1-methylimidazole/diethylenetriamine (Fluka, USA) (1:1 vol:vol)containing 50 μl of magnetic-hydroxylapatite beads (Chemicell, Germany).The mixture was gently inverted by rotation (LabQuake, USA) for 10minutes before the magnetic-hydroxylapatite beads were collected using apermanent magnet Promega, USA). The liquid was discarded and the beadswashed with 1 ml of 70% ethanol, collected with a magnet and washed with200 μl of water, collected with a magnet, the liquid discarded and thento the beads was added 200 μl of 10 mM EGTA pH 9.9 (NH4OH salt) elutionsolution and mixed for 4 minutes using a magnetic mixer (DexterMagnetics, UK). The beads were collected and the liquid containing theRNA transferred to a fresh tube, then a second batch of 200 μl of 10 mMEGTA pH 9.9 (NH4OH salt) elution solution was added to the beads, andfollowing 4 minutes mixing and bead collection, the liquid pooled withthe first elution and the RNA concentrated using a Microconultracentrifugation device as set out in example 1.

Alternatively, triethylenetetramine can be substituted fordiethylenetriamine in the deprotection reaction. The advantage oftriethylenetetramine is that it is less volataile and reactive comparedwith diethylenetriamine, however reaction times for completedeprotection may be slightly slower with the larger amine. Generally,amine bearing molecules which undergo substantial hydrogen bondingbetween molecules or have a larger molecular weight are less volatileand therefore preferred.

Alternatively, propionic (Fluka, USA), acrylic (Roth-Sochiel, Germany)or butyric anhydrides (Fluka, USA) can be subsituted for aceticanhydride in this example.

EXAMPLE 23 Deprotection of Acetyl Modified RNA Using Lysine or ArginineAqueous Solutions

To 200 μl of human plasma or serum containing the nucleic acid to bestabilised and purified was added 50 μl of 1-methylimidazole and brieflymixed. To this mixture was added 1 ml of N-methylpyrrolidinone/aceticanhydride (2:1 vol:vol), mixed briefly and incubated for 10 minutes at26-37° C. To the mixture was added 30 μl of a solution of 3.45M lysine,mixed and incubated for 2 min. at 26° C., then 50 μl ofmagnetic-hydroxylapatite Type I (Chemicell, Germany) was added and mixedfor 5 min. at 26° C. (MCB 1200, Dexter Magnetics, UK) to capture thenucleic acid. The magnetic-hydroxylapatite beads were collected with amagnet (Promega, USA) and briefly washed in 0.6 ml of 70% ethanol,before the beads were once again collected by magnet, the wash liquiddiscarded and 0.5 ml of a 3.45M Lysine solution or alternatively, 1 mlof a 1M Arginine solution was added and mixed for 10 minutes at 26° C.(MCB 1200, Dexter Magnetics, UK), the beads collected with a magnet andthe liquid discarded. The beads were then washed with 0.6 ml of 70%ethanol, the beads collected and the wash discarded and then washed with0.2 ml of water, the beads collected and the wash discarded followed byelution of the deprotected RNA using 0.2 ml of a 10 mM TEAH-EGTAsolution pH 10.1 with mixing for 5 min. (MCB 1200, Dexter Magnetics,UK). The eluted RNA can be separated from the remaining EGTA by forexample ultracentrifugation using a Microcon device as set out inexample 1.

EXAMPLE 24 Elution of RNA from Hydroxylapatite Using Ammonium Salts ofEGTA

It has been found that ammonium salts of EGTA and other chelators aremore effective at eluting nucleic acids from hydroxylapatite than sodiumor potassium salts. A 10 mM solution of EGTA was prepared by adding,dropwise, a 38% solution of NH4OH to a mixture of EGTA in water untilthe pH was brought to 9.9. This EGTA solution can be used as describedin example 22 as an elution solution.

EXAMPLE 25 Elution of RNA from Hydroxylapatite Using Tetra-AlkylAmmonium Salts of EGTA

It has been found that tetralkylammonium salts of EGTA and otherchelators are more effective at eluting nucleic acids fromhydroxylapatite than ammonium (NH3) salts. A 10 mM solution of EGTA wasprepared by adding, dropwise, a solution of either tetramethylammonium,tetraethylammonium or tetrabutylammonium hydroxide to a mixture of EGTAin water until the pH was brought to 9.9. It was found thattetraethylammonium-EGTA was marginally the most effective of the threetypes. This EGTA solution can be used as an alternative to the 10 mMEGTA pH 9.9 (NH4OH salt) elution solution as described in example 22.

EXAMPLE 26 Deprotection of Acetyl Modified RNA Using Amine ContainingDendrimers

To 20 μl of water containing 5 ng of acetylated BMV RNA was added 1 mgof a phospho-dendrimer-NH3 (Loup et al., (1999) Chem. Eur. J. 5:3644)and incubated for 13 minutes at 37° C. The dendrimer was removed bycentrifugation at 13 000 g for 30 seconds and the deprotected RNAanalysed. It was found that the dendrimer effectively removed the acetylgroups from the RNA.

EXAMPLE 26 Removal of Chelating Activity Using Photosensitive Chelators

RNA was isolated essentially as described in example 22 except the 10 mMEGTA pH 9.9 (NH4OH salt) elution solution was replaced with 10 mMDM-nitrophen (Calbiochem, USA) pH 8.0 elution solution. The elutionsolution containing the nucleic acid and the excess unwantedDM-nitrophen was subjected to photolysis essentially as described(Kaplan et al., (1985) Proc. Natl. Acad. Sci. 85:6571) in order toremove the chelating activity of the DM-nitrophen thereby leaving thechelator free RNA ready to be used in an appropriate reversetranscription-PCR assay.

EXAMPLE 27 Reduced Reagent Volumes

The ability to acetylate RNA using reduced volumes of solvent and aceticanhydride was examined. Acetylation was tested by the ability to modifya a 32P labelled RNA transcript, the percentage modification wasestimated using the altered mobility of the transcript in a denaturingsequencing gel as set out in WO/00/75302. To 200 μl of human plasma orserum containing the nucleic acid to be stabilised and purified wasadded 50 μl of 1-methylimidazole and briefly mixed. To this mixture wasadded 0.4 ml, 0.6 ml, 0.8 ml, 1 ml or 1.2 ml of dioxolane/aceticanhydride (2:1 vol:vol), mixed briefly, then 5 μl of 32P labelled RNAtranscript (Riboprobe, Promega, USA) and incubated for 10 minutes at 37°C. The 32P labelled RNA was then recovered from the reaction by adding50 μl of Type I hydroxylapatite beads (Chemicell, Germany), incubating 5min. with mixing, washing with 1 ml of 70% ethanol and eluting using 10mM EGTA pH9.9. The RNA was then loaded on a sequencing gel to determinethe extent of modification. It was found that the extent of RNAmodification was proportional to the volume of reagent added, exceptthat essentially the addition of 1 ml or 1.2 ml dioxolane/aceticanhydride (2:1) mixture led to the same amount of modification.Therefore the minimum amount of dioxolane/acetic anhydride (2:1) mixturenecessary for maximum acetylation was determined to be between 0.8-1 ml.It was found that using 0.6 ml of dioxolane/acetic anhydride (2:1)mixture that there was a small increase in the amount of acetylationbetween samples incubated at 37° C. for 1, 10 or 30 min. The amount ofacetylation also varied slightly according to the type of biologicalsample containing the RNA; RNA mixtures with human serum (Sigma-Aldrich,USA) being slightly less acetylated than human plasma or fetal calfserum.

EXAMPLE 28 Deprotection of Acetyl Modified RNA Following Removal of theReaction

To 200 μl of human plasma or serum containing the nucleic acid to bestabilised and purified was added 50 μl of 1-methylimidazole and brieflymixed. To this mixture was added 1 ml of N-methylpyrrolidinone/aceticanhydride (2:1 vol:vol), mixed briefly and incubated for 10 minutes at26-37° C. To the mixture was added 30 μl of a solution of 3.45M lysine,mixed and incubated for 2 min. at 26° C., then 50 μl ofmagnetic-hydroxylapatite Type I (Chemicell, Germany) was added and mixedfor 5 min. at 26° C. (MCB 1200, Dexter Magnetics, UK) to capture thenucleic acid. The magnetic-hydroxylapatite beads were collected with amagnet (Promega, USA) and briefly washed in 0.6 ml of 70% ethanol,before the beads were once again collected by magnet, the wash liquiddiscarded and 10-500 μl of diethylenetriamine was added to the wet beadsand mixed for 10 minutes at 26° C. (MCB 1200, Dexter Magnetics, UK), thebeads collected with a magnet and the liquid discarded. The beads werethen washed with 0.6 ml of 70% ethanol, the beads collected and the washdiscarded and then washed with 0.2 ml of water, the beads collected andthe wash discarded followed by elution of the deprotected RNA using 0.2ml of a 10 mM TEAH-EGTA solution pH 10.1 with mixing for 5 min. (MCB1200, Dexter Magnetics, UK). The eluted RNA can be separated from theremaining EGTA by, for example ultracentrifugation using a Microcondevice as set out in example 1.

EXAMPLE 29 Limiting Amounts of Elution Solution

It has been found that replacing the elution solution as set out inexample 22, with a single elution of 100 μl of 10 mM EGTA (NH4OH salt)pH 9.9 and an extended elution time of 30 minutes at 26° C. with mixing(MCB 1200, Dexter Magnetics, UK) is sufficient to remove a significantproportion of the RNA from the magnetic-hydroxylapatite withoutsignificantly contaminating the eluted RNA with EGTA, so the RNA can beused directly without further removal steps of the EGTA. Effectively allthe EGTA added is effectively bound to the magnetic-hydroxylapatite.

EXAMPLE 30 Stability of Mixtures of Reagents

The stability of a mixture of anhydrides with various solvents andcatalysts was examined. The activity of the acetic anhydride afterstorage for 11 days at 26° C. was tested by the ability to modify a a32P labelled RNA transcript, the percentage modification was estimatedusing the altered mobility of the transcript in a denaturing sequencinggel as set out in WO/00/75302. It was found that the following mixtureswere active after storage for 11 days at 26° C.;N-methylpyrrolidinone/acetic anhydride/1-methylimidazole (2:1:0.15,vol:vol:vol), N-methylpyrrolidinone/propionicanhydride/1-methylimidazole (2:1:0.15, vol:vol:vol),N-methylpyrrolidinone/acetic anhydride/4-pyrrolidinopyridine (2:1:0.15,vol:vol:vol). Intermediate activity was determined for the mixtureN-methylpyrrolidinone/propionic anhydride/4-pyrrolidinopyridine(2:1:0.15, vol:vol:vol), and essentially inactive mixtures wereN-methylpyrrolidinone/acetic anhydride/2-hydroxypyridine (2:1:0.15,vol:vol:vol) and N-methylpyrrolidinone/aceticanhydride/2-hydroxypyridine (2:1:0.15, vol:vol:vol). In these examples,the solvent N-methylpyrrolidinone can be replaced by either formylmorpholine or dimethyl imidazolidone. Mixtures of solvent, aceticanhydride and 1-methylimidazole turned from a clear to a dark browncolour after 1 hr at room temperature, whilst this colour change wasless apparent in propionic anhydride containing mixtures.

EXAMPLE 31 Reactions Using Other Catalysts

To 200 μl of human plasma or serum containing the nucleic acid to bestabilised and purified was dissolved 50 mg of either4-pyrrolidinopyridine or 2-hydroxypyridine catalysts instead of1-methylimidazole. To this mixture was added 1 ml ofN-methylpyrrolidinone/acetic anhydride (2:1 vol:vol), mixed briefly andincubated for 10 minutes at 26-37° C. The RNA was then deprotected andpurified according to the method of example 22. It was found that either4-pyrrolidinopyridine or 2-hydroxypyridine catalysts could substitutefor 1-methylimidazole.

Alternatively, propionic (Fluka, USA), acrylic (Roth-Sochiel, Germany)or butyric anhydrides (Fluka, USA) can be substituted for aceticanhydride in this example.

EXAMPLE 31 Protection of Modified RNA from Freeze-Thaw Degradation

Preparation of modified RNA. To 40 μl of tetrahydrofuran containing 16%1-methylimidazole was added 100 ng-1 μg of BMV RNA (Promega, USA), then2-4 μl of acetic anhydride was mixed into the reaction and incubated for10 minutes at 37° C. Following ethanol precipitation of the modified RNAand resuspension in 40 μl of water, an aliquot of both modified andnon-modified BMV RNA was put in separate 2 ml polypropylene tubes andfrozen in a mixture of dry-ice ethanol for 20 seconds followed bythawing at 42° C. for 30 seconds. This cycle was repeated ten times intotal. The RNA was mixed with 50% formamide and loaded on a 1.2% agarosegel. Whilst the unmodified RNA was significantly degraded, the modifiedRNA showed no signs of degradation by freeze thawing.

EXAMPLE 32 Extraction of HCV Viral RNA from Clinical Samples; 1.45 mlReaction Volume

Stabilisation of the RNA analyte: To 200 μl of human plasma (EDTAcoagulation inhibitor) or serum in a 15 ml screw top polypropylenecentrifuge tube (Falcon, USA) was added 50 μl of 1-methylimidazole, themixture mixed briefly and then 600 μl of a mixture of tetrahydrofuranand acetic anhydride (2:1 vol/vol) was added and mixed by gentlepipetting with a 1 ml pipette tip. A second addition of 600 μl oftetrahydrofuran and acetic anhydride (2:1 vol/vol) was added within 1minute of the first addition and the solution mixed again.

Following addition of the acylation reagent, the internal control (8 μlof the HCV Internal Control, version 2.0, Roche Diagnostics, USA) isadded within 15 seconds. Following an incubation of 10 minutes at 37°C., the solution containing the stabilised RNA can be stored forprolonged periods at up to 37° C.

To 1.45 ml of the reaction containing the desired RNA analyte is added1.4 ml of ice cold 1-methylimidazole and the solution mixed by gentlepipetting. Then three separate aliquots of 200 μl of a prepared solutionof 1 ml of 1-methylimidazole, 1 ml of ethylenediamine containing 50 μl(40 mg/ml) of phosphate treated hydroxylapatite Type I (Chemicell,Germany) are added and the reaction incubated for 2 minutes at 25° C.,before the remaining 1.45 ml of the 1-methylimidazole, primary amine andbeads are added and mixed. The entire mixture is then slowly mixed usingan end-over-end wheel for 10 minutes at 25° C. to allow deprotection ofthe RNA and binding to the beads. However, agitating the mixture is notessential.

The beads are then collected from the reaction using a magnetic stand(Dynal, Norway) and the liquid discarded by pouring it away from thebead pellet. The beads are then resuspended in 1 ml of 70%methanol/ethanol (1:1 vol/vol) and transferred to a fresh 2 mlpolypropylene centrifuge tube and collected using a magnetic stand(Promega, USA). The beads are washed two more times in 1 ml of 70%methanol/ethanol and then washed three times in 100 μl of water. It isimportant not to touch the beads with the pipette tip when they are inaqueous suspension because they tend to stick to plastic resulting insample loss. The RNA is now ready for storage, transport or can beimmediately eluted from the hydroxylapatite beads.

To the nucleic acid hydroxylapatite bead mixture (approximate volume50-100 μl) is added 50 μl of a 10 mM EGTA (NaOH buffered) solution (pH8), the beads agitated by means of a magnetic stirrer set on the program0.5 second step, (MCB 1200, Dexter Magnetics, UK) for 2 minutes at 25°C., the beads collected using the magnet, the liquid collected and thenthe process repeated 7 more times and the liquid containing the nucleicacid pooled.

The preferred method of filtration for concentration and removal of EGTAis as follows. Using 10 mM EGTA as the elution solution, the first 400μl of the elution from the hydroxylapatite beads is collected andpooled. The elution can either be 8 separate elutions of 50 μl with 2minute elution steps using mixing as described above (MCB1200, DexterMagnetics, USA) or 1 elution with 400 μl of 10 mM EGTA with a ten minuteelution with mixing. It is also possible to add a smaller volume of moreconcentrated elution solution such as μl of 20 mM EGTA and mixing for 10minutes at room-temperature. In any case, the final elution volume isconveniently no more than 400 μl which is the maximum volume accomodatedby the Microcon filtration device (Part No. 42416, Millipore, USA). Thedevice is then centrifuged at 12 000 g for 20 minutes (until dryness),400 μl of water added and the device spun again at 12 000 g for 20minutes (until dryness). Then 25-50 μl of water is added to the deviceto recuperate the nucleic acid the cup inverted in a 2 ml fresh tube andcentrifuged for 10 seconds at 2500 g. The nucleic acid can then be usedin the assay. Proteins that are larger than 50 000 daltons are alsoretained with the nucleic acid, however, it has been found that nodetectable inhibition occured during RT-PCR(HCV Amplicor v2.0, RocheDiagnostics, USA) with RNA prepared in this manner from blood plasma.

It has been found that HCV viral RNA can be detected at 300 copies perml (as determined by Roche Amplicor Monitor version 1.0) of plasma usingthis stabilisation and purification method (see table 2). TABLE 2Summary of results obtained using MRT (method as described in example32) compared with Roche purification (Amplicor v2.0). S/CO representsthe value of the OD 660 nm reading divided by the OD 660 nm cut-offvalue of 0.15. Copies/ml MRT Roche (Amplicor MRT Roche PurificationPurification Monitor HCV Purification Purification Incubation 37° C.*Incubation 37° C.* Identification Génotype Version 1.0) ParticularitésS/CO S/CO S/CO S/CO ETS.2 1a 2 500 ≧26.67 24.60 0.41 0.04 ETS.4 2a/2c300 16.27 8.00 0.71 0.033 ETS.7 1 40 000 000 25.8 25.73 ≧26.67 ≧26.67ETS.10 3a <seuil 2.13 1.67-9.33-19.33 0.033 0.04 ETS.12 5a 500 ≧26.6710.2 0.027 0.04 ETS.21 1b 306 800 24.64 17.27 ≧26.67 25.81 ETS.25 5a 3300 ≧26.67 22.67 16.21 0.046 ETS.26 1a 422 700 Anti HCV (−) 25.81 24.6625.8 25.81 ETS.32 3a 69 500 +HBV ≧26.67 25.8 8.63 0.046 ETS.34 1b 364000 +HGV 25.81 24.67 22.63 10.65 Run control 1b / 50I U/ml 21.15 / 0.8 /

EXAMPLE 33 Extraction of HCV Viral RNA from Clinical Samples Followingan Incubation at 37° C. for One Week

To test the stabilising activity of modifying RNA, HCV positive plasmasamples were mixed with the reagents as set out in example 32, but priorto the addition of 1.4 ml of ice cold 1-methylimidazole, the sampleswere incubated at 37° C. for one week. Following the incubation, thepurification of the HCV RNA samples was continued from the step ofadding 37° C. for one week as set out in example 32. HCV RNA wasdetected using HCV Amplicor v2.0, (Roche Diagnostics, USA) and comparedwith HCV RNA incubated at 37° C. for one week in Roche Amplicor Lysissolution before standard Amplicor v2.0 purification. Results are shownin Table 2. Stability using the method in example 32 (column entitled<<MRT purification incubation at 37° C.>> in Table 2) was at least asgood as that provided by the chaotrope guanidine in the Roche lysisbuffer.

EXAMPLE 33 Testing HCV Negative Plasma Samples

Seven HCV negative plasma samples were tested using the method as setout in example 32 and RNA tested with HCV Amplicor v2.0, (RocheDiagnostics, USA). One sample contained added genomic DNA and anotherwas HGV positive. All seven samples were negative for HCV but positiveas expected for the internal control demonstrating the specificity ofdetection.

1. A method for the stabilisation of nucleic acid from a biologicalsample, which comprises: (a) collecting a biological sample; (b)treating the sample so that a proportion of the 2′, 3′ or 5′-OHpositions of the nucleic acid are modified with a protecting group; and(c) subjecting the treated sample to one or more steps to isolatenucleic acid therefrom; wherein the modified nucleic acid is subjectedto a deprotection step comprising treatment with a primary amine toremove the protecting group.
 2. The method according to claim 1, whereinthe biological sample comprises viruses, cells, body fluids, blood,serum or plasma.
 3. The method according to claim 1, wherein thebiological sample comprises a clinical sample or a human pathogen. 4.The method according to claim 1, wherein the nucleic acid is single ordouble stranded RNA or DNA.
 5. The method according to claim 4, whereinthe sample is treated with a reactant capable of covalently modifyingthe 2′-OH position of the ribose rings of the RNA.
 6. The methodaccording to claim 1, wherein step (b) is carried out in the presence ofan organic solvent.
 7. The method according to claim 6, wherein theorganic solvent has a flashpoint above 37° C.
 8. The method according toclaim 6, wherein the organic solvent is capable of forming a homogeneoussolution with human blood when mixed in a ratio of 5:1 (vol:vol).
 9. Themethod according to claim 1, wherein the primary amine isethylenediamine, diethylenetriamine, triethylenetetramine, lysine orarginine.
 10. The method according to claim 1, wherein step (c)comprises: (i) binding the nucleic acid to a solid phase; (ii)optionally washing the solid phase to remove contaminants; and (iii)optionally eluting the nucleic acid from the solid phase.
 11. The methodaccording to claim 10, wherein the solid phase comprises magneticparticles.
 12. The method according to claim 10, wherein the solid phasecontains a metal or metal ion capable of coordinating with phosphate.13. The method according to claim 12, wherein the nucleic acid is elutedwith a chelator.
 14. The method according to claim 13, wherein thechelator is EGTA and elution is carried out at a pH above
 9. 15. Themethod according to claim 13, wherein the chelator is a salt of ammoniaor tetra-alkylammonium.
 16. The method according to claim 13, whichfurther comprises removing the chelator from the nucleic acid byultrafiltration, photosensitivity of the chelator or affinitypurification using an affinity tag on the chelator.
 17. The methodaccording to claim 12, wherein the solid phase compriseshydroxylapatite.
 18. The method according to claim 17, wherein thehydroxylapatite is pretreated with a phosphate-containing compound. 19.The method according to claim 17, wherein the hydroxylapatite is washedin step (ii) with an amine.
 20. The method according to claim 19,wherein the amine is a primary amine.
 21. The method according to claim20, wherein the deprotection step comprises step (ii).
 22. The methodaccording to claim 9, wherein the deprotection step occurs between step(i) and step (ii).
 23. The method according to claim 10, wherein thesolid phase comprises silica.
 24. The method according to claim 10,wherein the solid phase has immobilised thereon nucleic acidcomplementary to the nucleic acid targeted for isolation.
 25. The methodaccording to claim 24, wherein the nucleic acid targeted for isolationis RNA, which is subjected to the deprotection step prior to binding tothe solid phase.
 26. A kit for use in a method for the stabilisation ofnucleic acid from a biological sample; which comprises: (i) a reactionsystem for treating the sample so that a proportion of the 2′, 3′ or5′-OH positions of the nucleic acid are modified with a protectinggroup; (ii) an isolation system for subjecting the treated sample to oneor more steps to isolate nucleic acid therefrom; and (iii) a primaryamine for subjecting the modified nucleic acid to a deprotection step toremove the protecting group.
 27. The kit according to claim 26, whereinthe reaction system comprises a reactant capable of covalently modifyingthe 2′-OH position of the ribose rings of RNA.
 28. The kit according toclaim 26, wherein the reaction system includes an organic solvent. 29.The kit according to claim 28, wherein the organic solvent has aflashpoint above 37° C.
 30. The kit according to claim 28, wherein theorganic solvent is capable of forming a homogeneous solution with humanblood when mixed in a ratio of 5:1 (vol:vol).
 31. The kit according toclaim 26, wherein the primary amine is ethylenediamine,diethylenetriamine, triethylenetetramine, lysine or arginine.
 32. Thekit according to claim 26, wherein the isolation system comprises: (a) asolid phase for binding the nucleic acid; (b) optionally a washingsolution for washing the solid phase to remove contaminants; and (c)optionally an elution solution for eluting the nucleic acid from thesolid phase.
 33. The kit according to claim 29, wherein the solid phasecomprises magnetic particles.
 34. The kit according to claim 32, whereinthe solid phase contains a metal or metal ion capable of coordinatingwith phosphate.
 35. The kit according to claim 34, wherein the elutionsolution comprises a chelator.
 36. The kit according to claim 35,wherein the chelator is EGTA.
 37. The kit according to claim 35, whereinthe chelator is a salt of ammonia or tetra-alkylammonium.
 38. The kitaccording to claim 35, wherein the chelator is a photosensitive chelatoror has an affinity tag.
 39. The kit according to claim 34, wherein thesolid phase comprises hydroxylapatite.
 40. The kit according to claim39, wherein the hydroxylapatite is pretreated with aphosphate-containing compound.
 41. The kit according to claim 39,wherein the washing solution comprises an amine.
 42. The kit accordingto claim 41, wherein the amine is the primary amine.
 43. The kitaccording to claim 32, wherein the solid phase comprises silica.
 44. Thekit according to claim 32, wherein the solid phase has immobilisedthereon nucleic acid complementary to the nucleic acid targeted forisolation.